Imaging of the Vulnerable Atherosclerotic Plaque. Pre-Clinical Evaluation of PET Tracers for Vascular Inflammation

Atherosclerosis is a vascular inflammatory disease causing coronary artery disease, myocardial infarct and stroke, the leading causes of death in Finland and in many other countries. The development of atherosclerotic plaques starts already in childhood and is an ongoing process throughout life. Rupture of a plaque and the following occlusion of the vessel is the main reason for myocardial infarct and stroke, but despite extensive research, the prediction of rupture remains a major clinical problem. Inflammation is considered a key factor in the vulnerability of plaques to rupture. Measuring the inflammation in plaques non-invasively is one potential approach for identification of vulnerable plaques. The aim of this study was to evaluate tracers for positron emission tomography (PET) imaging of vascular inflammation. The studies were performed with a mouse model of atherosclerosis by using ex vivo biodistribution, autoradiography and in vivo PET and computed tomography (CT). Several tracers for inflammation activity were tested and compared with the morphology of the plaques. Inflammation in the atherosclerotic plaques was evaluated as expression of active macrophages. Systematic analysis revealed that the uptake of 18F-FDG and 11C-choline, tracers for metabolic activity in inflammatory cells, was more prominent in the atherosclerotic plaques than in the surrounding healthy vessel wall. The tracer for αvβ3 integrin, 18Fgalacto- RGD, was also found to have high potential for imaging inflammation in the plaques. While 11C-PK11195, a tracer targeted to receptors in active macrophages, was shown to accumulate in active plaques, the target-to-background ratio was not found to be ideal for in vivo imaging purposes. In conclusion, tracers for the imaging of inflammation in atherosclerotic plaques can be tested in experimental pre-clinical settings to select potential imaging agents for further clinical testing. 18F-FDG, 18F-galacto-RGD and 11C-choline choline have good properties, and further studies to clarify their applicability for atherosclerosis imaging in humans are warranted.Siirretty Doriast

Tools for the Advancement of Radiopharmaceutical Therapy

Radiopharmaceutical therapy is used to treat cancers and other diseases with radiolabeled pharmaceuticals. The treatment targets specific cells, and the emitted ionizing radiation cause cytotoxic damage. Dosimetry is performed to estimate the absorbed dose from the energy deposited in the body. This requires measurement of the activity in vivo and knowledge of the retention time of the activity in tumor and organs. Preclinical trials precede clinical studies and evaluate the potential of new radiopharmaceuticals for treatment. Similarly, in vitro and in vivo experiments with radiopharmaceuticals and sources of ionizing radiation are performed to increase radiobiological knowledge, which is helpful in the optimization of radiopharmaceutical therapy. Dosimetry is also necessary for these studies to correctly quantify the biological response to ionizing radiation.However, standard dosimetry only considers macroscopic volumes such as organs or solid tumors. Due to the short range of the emitted radiation, heterogeneous activity uptake can generate heterogeneous energy depositions. In a tumor, this means a large variation in particle tracks hitting the cell nuclei, where cells inundertreated areas will not receive any particle tracks through the cell nucleus. Since damage to DNA in the cell nucleus is the main cause of radiation-induced cell death, this can reduce the treatment effect. Early insight into these limitations of a new radiopharmaceutical can be achieved in preclinical studies investigatingthe intra-tumoral distribution of the radiopharmaceutical uptake. Paper 4 investigated the tumor control probability from the intra-tumoral distribution of 177Lu-PSMA-617 in LNCaP xenografts. Monte Carlo simulations can be used for small-scale and microscopic dosimetry, where small targets such as cells and cellnuclei are considered. Similarly, in paper 3, simulations of an alpha particle source and cell nuclei irradiated were used to estimate the distribution of induced γ-H2AX foci in PC3 cells irradiated with an 241Am source in vitro.In preclinical studies of therapeutic radiopharmaceuticals, xenografted animal models are followed postinjection over long periods to evaluate the treatment response. This is usually done by measuring changes in tumor size over time. In addition, molecular imaging with positron emission tomography (PET) offers anopportunity to measure biochemical changes in vivo, such as the radiation damage response. However, as investigated in paper 1, gamma emission from the therapeutic radiopharmaceutical in the animal model can cause perturbations to the image by increasing dead-time losses and causing signal pile-up. However, assuggested in paper 2, preclinical intra-therapeutic PET imaging can still be performed during 177Lu-labeled radiopharmaceutical therapy, with shielding attenuating the excess photons while still allowing coincidence detection of annihilation photons

Investigation of timepix radiation detector for autoradiography and microdosimetry in targeted alpha therapy

The Timepix detector developed by CERN is a novel and sophisticated particle detector. It consists of a semiconductor layer divided into an array of pixels. This array of pixels is bumpbonded to an electronics integrated layer (i.e. the readout chip). Timepix can be used for a wide range of measurements of electromagnetic radiation and particles and their applications in different fields such as space physics, nuclear physics, radiotherapy physics, imaging and radiation protection. The Timepix detector used in this work was purchased from Amsterdam Scientific Instruments, the Netherlands, in order to investigate its use for microdosimetry purposes, in particular in targeted alpha therapy. The device has the following properties: 256 x 256 pixels of 55 x 55 μm2 area each, the chip is effective for positive or negative charge and can be used to detect electrons, X-rays, neutrons and heavy charge particles. It can work as an energy spectrometer, has good spatial resolution and reason1ble detection efficiency. The device can operate in three common modes: Timepix mode, Medipix mode, and Time-Over-Threshold (TOT) mode. Targeted alpha therapy (TAT) is a novel type of radionuclide therapy in which an alpha emitting radioisotope is attached to a cancer cell seeking vector (so called radioimmunoconjugate (RIC)). Once attached to a cancer cell, it causes localized damage due to traversal and energy deposition high LET a-particles. There is, however, a lack of data related to a-particle distribution in TAT. These data are required to more accurately estimate the absorbed dose on a cellular level. As a result, this work aims to develop a microdosimetry technique, using Timepix detector that will estimate, or better yet determine the absorbed dose deposited by a-particles in cells as well as will measure the biodistribution of the radioisotope in a tumour. Initially, extensive Timepix characterization and testing has been done to evaluate the detector's response, including linearity, reproducibility, and sensitivity to low doses of radiations (μGy-mGy dose region) and energy dependence. 1-125 seeds and superficial X-rays (below 70 kVp), produced by the Gulmay superficial X-ray unit, were used. The measured Timepix pixel value was correlated with the known dose (based on the irradiation time used and TLD-100 measurements) and a pixel-value-to- dose calibration curve was obtained. It was confinned that Timepix value increased linearly with the dose delivered. The dose calibration curves using the superficial X-ray beams showed that the pixel value, however, depended on the energy of the X-ray beam. The application of Timepix to measure radioisotope biodistribution (i.e. autoradiography) was investigated. Mice with Lewis lung (LL2) tumours were treated with about 18 kBq oP27Thlabelled DAB4 murine monoclonal antibody that bounds to necrotic tumour cells. The rationale is to develop a-particle-mediated bystander kill of nearby viable tumour cells. To generate more necrotic tumour cells for 227Th-DAB4 binding, some mice also received chemotherapy before being injected with Th-227-DAB4. Finally, 5 mm tumour sections were cut from treated mice for autoradiography with Timepix. Each tumour section was mounted onto a slide with front face uncovered to allow emission of a-particles from the tumour section. Simple steel collimator (I cm radius, 2 cm length) was manufactured in-house and positioned around the tumour section. The slide was placed 2 cm away from the Timepix detector. Bias voltage of 7 V was applied, and a-particle filter was selected for acquisition. Detector cover was removed, exposing the Si layer, to allow the emitted a-particles ( - 6 Me V) to reach the detector. Image acquisition took -14 h. Good resolution autoradiographs of radiolabelled tumour sections were acquired, showing a-particle, electron and X-ray tracks. Timepix measurements also showed an increased Th-227-DAB4 uptake following chemotherapy due to increase in necrotic tissue volume. Timepix was also used to measure the uptake of Cr-51 by A549 cells (lung carcinoma cell line) for different pH levels and the dependence of uptake on pH was investigated. Timepix was observed to be sensitive to detect small changes in the activity/uptake of radioactive sources depending on the environmental condition and the number of cells. The last part of this thesis deals with the development of a transmitted a-particle microdosimetry technique. First, A549 cells were grown in vitro using standard protocols and were irradiated using a 6 MY photon beam with different doses varying between 2-8 Gy and Ra-226 source was used for a-particle irradiation to evaluate A549 radiation sensitivity using clonogenic assay and MTT assay. The cell line was found radiosensitive, with 050 of~ 2 Gy for X-ray irradiation. For transmitted dosimetry, A549 cells were either unirradiated (control) or irradiated for ~2, 1, 2 or 3 hours with a-particles emitted from a Ra-223 source positioned below a monolayer of A549 cells. The HTS Transwell" 96 well system (Corning, USA), consisting of 2 compartments, was used to develop a method for tracking a-particles through a cell mono layer. This system comprises of two compartments, with liquid Ra-223 evaporated in the lower compartment to avoid a-particle self-absorption inside the liquid. The measured activity of 5 kBq was unifonnly distributed, as confirmed by Timepix detector. The second compartment consists of a flat bottom polycarbonate membrane (I 0 μm thick) where cells are plated. It is sufficiently thin to allow a-particles to penetrate through and hit the cells. Fifteen thousand A549 cells were seeded in the upper compartment that was then inserted into the lower compartment containing the evaporated Ra-223. The transwell system was positioned under the Timepix detector. Transmitted a-particles were detected for 1;2, I, 2 or 3 hour irradiation times. Additionally, DNA double strand breaks (DSBs) in the form of y-H2AX foci, were examined by fluorescence microscopy. The number of transmitted a-particles was correlated with the observed DNA DSBs and the delivered radiation dose was estimated. Additionally, the dose deposited was calculated using Monte Carlo code SRIM. Approximately 20% of a-particles were transmitted and detected by Timepix. The frequency and number of y-H2AX foci increased significantly following a-particle irradiation as compared to unirradiated controls. The RBE equivalent dose delivered to A549 cells was estimated to be approximately 0.66 Gy, 1.32 Gy, 2.53 Gy and 3. 96 Gy after Y2, I, 2 and 3 h irradiation, respectively, considering a relative biological effectiveness of a-particles of 5.5. In summary, the Timepix detector can be used effectively for autoradiography in TAT, providing high resolution images and excellent spatial resolution of detected a-particles, as well as a transmitted a-particle microdosimetry detector. If cross-calibrated using biological dosimetry, this method will give a good indication of the biological effects of a-particles without the need for repeated biological dosimetry which is costly, time consuming and not readily available.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Physical Sciences, 2017

Development of Radiotracers for Neuroimaging

Nuclear imaging enables quantitative measurements of biological processes in vivo and has revolutionised biomedical research, drug development and clinical practice. Despite the advances made in this field, the ability to image fundamental aspects of neurological diseases remains a challenge. This is partly due to the limited availability of radiotracers for imaging excitatory neurotransmission and detection of inflammation as well as an array of other biochemical processes central to the operational function of the brain. The aim of this research was to expand the arsenal of radiotracers available for neuroimaging in order to study key pathological processes involved in neurological diseases. With the aim to target neuronal Voltage Gated Sodium Channels (VGSCs), Vascular Cell Adhesion Molecule – 1 (VCAM-1) and N-methyl-D-Aspartate Receptors (NMDARs), radiotracers have been synthesised and evaluated. Abnormal expression of these receptors has been implicated in a number of pathological conditions including epilepsy, multiple sclerosis and neurodegeneration. The radiotracers were characterised and evaluated via in vivo imaging (MRI and SPECT/CT) and ex-vivo studies (phosphorimaging, biodistribution and metabolite analysis) in order to determine if they hold significant potential as tools to study neuronal pathways as well as for diagnostic imaging and treatment monitoring. Iodinated analogues of the iminodihydroquinoline WIN17317-3, and the 1-benzazepin-2-one BNZA have been evaluated as neuronal VGSC tracer candidates in healthy mice. Whilst the WIN17317-3 analogue suffered from poor brain uptake and was rapidly metabolised in vivo, the BNZA analogue exhibited excellent in vivo stability and its promising uptake in the brain warrants further investigations. Even though N-(1-Napthyl)-N’-(3-[123I]-iodophenyl)-N’-methylguanidine ([123I]CNS-1261) has demonstrated favourable pharmacokinetics for brain imaging in clinical studies, [125I]CNS-1261 was not successful in discriminating NMDAR expression between naïve rats and those induced with status epilepticus using lithium and pilocarpine. Promisingly, a multi modal contrast agent comprising micron sized particles of iron oxide conjugated to I-125 radiolabelled antibodies, highlighted the up-regulation of VCAM-1 in rat models of cerebral inflammation and in the lithium pilocarpine model of status epilepticus. This versatile imaging agent presents an exciting opportunity to identify an early biomarker for epileptogenesis

NEPTUNE (Nuclear process-driven Enhancement of Proton Therapy UNravEled)

Protontherapy is an important radiation modality that has been used to treat cancer for over 60 years. In the last 10 years, clinical proton therapy has been rapidly growing with more than 80 facilities worldwide [1]. The interest in proton therapy stems from the physical properties of protons allowing for a much improved dose shaping around the target and greater healthy tissue sparing. One shortcoming of protontherapy is its inability to treat radioresistant cancers, being protons radiobiologically almost as effective as photons. Heavier particles, such as 12C ions, can overcome radioresistance but they present radiobiological and economic issues that hamper their widespread adoption. Therefore, many strategies have been designed to increase the biological effectiveness of proton beams. Examples are chemical radiosensitizing agents or, more recently, metallic nanoparticles. The goal of this project is to investigate the use of nuclear reactions triggered by protons generating short-range high- LET alpha particles inside the tumours, thereby allowing a highly localized DNA-damaging action. Specifically, we intend to consolidate and explain the promising results recently published in [2], where a significant enhancement of biological effectiveness was achieved by the p-11B reaction. Clinically relevant binary approaches were first proposed with Boron Neutron Capture Therapy (BNCT), which exploits thermal neutron capture in 10B, suitably accumulated into tumour before irradiation. The radiosensitising effects due to the presence of 10B will be compared to those elicited by p-11B, using the same carrier and relating the observed effects with intracellular 11B and 10B distribution as well as modelled particle action and measured dose deposition at the micro/nanometer scale. Moreover, the p-19F reaction, which also generates secondary particles potentially leading to local enhancement of proton effectiveness, will be investigated. The in-vivo imaging of 11B and 19F carriers will be studied, in particular by optimizing 19F-based magnetic resonance

Multi-scale dosimetry for targeted radionuclide therapy optimisation

La Radiothérapie Interne Vectorisée (RIV) consiste à détruire des cibles tumorales en utilisant des vecteurs radiomarqués (radiopharmaceutiques) qui se lient sélectivement à des cellules tumorales. Dans un contexte d'optimisation de la RIV, une meilleure détermination du dépôt d'énergie dans les tissues biologiques est primordiale pour la définition d'une relation dose absorbée - effet biologique et pour l'optimisation des traitement du cancer. Cela nécessite une évaluation quantitative de la distribution de l'activité (avec la technique d'imagerie moléculaire la plus appropriée) et d'effectuer le transport du rayonnement à l'échelle à laquelle se produisent les phénomènes biologiques pertinents. Les méthodologies à appliquer et les problématiques à établir dépendent strictement de l'échelle (cellule, tissu, organe) de l'application considérée, et du type de rayonnement en cause (photons, électrons, particules alpha). Mon travail de recherche a consisté à développer des techniques dosimétriques dédiées (dosimétrie mono-échelle) et innovantes, capables de prendre en compte la particularité de différents scénarios expérimentaux (cellulaire, pré-clinique, RIV clinique).Targeted Radionuclide Therapy (TRT) consists in killing tumour targets by using radiolabeled vectors (radiopharmaceuticals) that selectively bind to tumour cells. In a context of TRT optimization, a better determination of energy deposition within biologic material is a prerequisite to the definition of the absorbed dose-effect relationship and the improvement of future cancer treatment. This requires being able to quantitatively assess activity distribution (with the most appropriate molecular imaging technique) and perform radiation transport at the scale at which biologically relevant phenomena occur. The methodologies that should be applied and the problematic to be faced strictly depend on the scale (cell, tissue, body) of the application considered, and on the type of radiation involved (photons, electrons, alpha). This research work consisted in developing dedicated dosimetric techniques (single-scale dosimetry) capable of taking into account the peculiarity of different experimental scenarios (cellular, pre-clinical, clinical TRT)

Radio-pathological Investigation of Tau Tracers in Neurodegenerative Diseases

Abnormal accumulation of misfolded tau protein is a major hallmark of pathology underlying multiple neurodegenerative tauopathies, of which Alzheimer’s disease (AD) is the most common. Tau burden in AD correlates with cognitive decline and has consequently been proposed as a major biomarker of disease progression. The development of tau-specific ligands for imaging with positron emission tomography (PET) holds immense promise in accelerating clinical diagnoses of AD, and other dementias. Two distinct generations of tau PET radiotracers are at different stages of development and have been rapidly translated into clinical studies. However, the neuropathological validation of these tracers remains limited and concerns have been raised over low sensitivity and off-target binding, particularly for first-generation tau ligands. The correct interpretation of clinical PET scans requires greater understanding of tracer binding profiles at both the molecular and cellular level. Human post-mortem brain tissue provides a robust platform to validate the binding profiles of tau tracers. In this thesis, the binding profiles of structurally distinct first- (T726/Flortaucipir, THK-5117) and second-generation (PI-2620, MK-6240) tau tracers were characterised, with the ultimate aim to inform on their clinical applications. The sensitivity and specificity of tau tracers were examined using human post-mortem brain tissue from cases with AD, primary tauopathies, non-tau proteinopathies and in age-matched controls. Tracers from both generations were found to selectively depict paired-helical filament tau and therefore suited to differentiate AD from controls and non-AD dementias. Second-generation tracers had greater sensitivity to depict tau in early disease than first-generation tracers. Overall, second-generation tau radiotracers have improved upon earlier compounds, including overcoming limitations associated with off-target binding. However, for familial primary tauopathies there is currently limited clinical use for tau radiotracers. Further investigation is required to understand the clinical potential of tau PET tracers for their use to diagnose sporadic primary tauopathies

Cyclotron Production and Biomedical Imaging Applications of the PET Isotope Manganese-52

Manganese is an important element for biomedical research because of its roles as an essential micronutrient and as a neurotoxin from chronic elevated exposure, as well as the role of manganese(II) as a paramagnetic core for contrast agents in T1-weighted magnetic resonance imaging (MRI). Using a radiotracer of manganese provides excellent sensitivity for studying these phenomena, but only 52Mn met the criteria for our experiments: (1) a half-life (t1/2=5.6 days) that was long enough to examine timepoints over several days, (2) a half-life that was short enough to emit sufficient counts for a realistic scan time, and (3) emitted radiation of a variety and energy that were appropriate for existing pre-clinical imaging modalities. Manganese-52 is well-suited for imaging with positron emission tomography (PET) because it emits positrons with a low energy (E+=242 keV), which improves spatial resolution, and with an acceptable total abundance for positron emission (I+=29.6%) for adequate signal. Manganese-52 was produced on site by the 52Cr(p,n)52Mn reaction by bombarding non-enriched chromium (52Cr: 83.8%) with ~13 MeV protons that were accelerated in the CS-15 cyclotron at Washington University School of Medicine in St. Louis. Bombardments of stacks of thin chromium metal foils were used to measure nuclear cross-sections for the natCr(p,x)52,52m,54Mn reactions, with results that agreed closely to simulations and published results. Manganese-52 was separated chemically from bombarded chromium metal by cation- or anion-exchange chromatography. The separated product was used in experiments that included biodistribution by injection or inhalation, PET/CT or PET/MR in phantoms and rodents, and radiolabelling of a Mn(II)-based contrast agent for T1-weighted MRI. To reduce radiation dose to production personnel, we designed a remotely controlled, semi-automated module for the remote separation of 52Mn inside a lead hot cell. This module was similar to other modules that we designed, built, and tested for the routine, scaled-up production of larger quantities of the PET isotopes 89Zr and 86Y. We anticipate that the module for 52Mn will be completed, routine production of greater quantities of 52Mn will be achieved, and this radioisotope will continue to be used to study and image the interesting aspects and behaviors of manganese chemistry