Development of novel radiotracers as tools for imaging the human brain

Introduction: Brain imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to study the processes underlying neurological and psychiatric disorders. In addition, in vivo brain imaging using SPECT or PET may provide new approaches for drug target identification, pre-clinical testing and occupancy studies, and therefore improve drug discovery. The utility of in vivo brain imaging using SPECT or PET relies on the ability of different radiotracers (typically organic compounds labelled with radionuclides) to bind to a wide variety of targets, including receptors, transporters and enzymes. Therefore the development of novel radiotracers for in vivo brain imaging using SPECT of PET is of vital importance. This thesis is focused on the process of developing novel radiotracers as tools for imaging the human brain, where the radiotracer discovery and development pipeline is discussed and each step prior to clinical trials investigated. Radiotracer discovery: Previously, discovery of novel brain radiotracers has largely relied on simplistic screening tools. Improved selection methods at the early stages of radiotracer discovery and an increased understanding of the relationships between in vitro physicochemical and in vivo radiotracer properties are needed. This thesis investigated if high performance liquid chromatography (HPLC) methodologies could provide criteria for lead candidate selection by comparing HPLC measurements with radiotracer properties in humans. In this study, ten molecules, previously used as radiotracers in humans, were analysed to obtain the following measures: partition coefficient (Log P); permeability (Pm); percentage of plasma protein binding (%PPB); and membrane partition coefficient (Km). Relationships between brain entry measurements (Log P, Pm and %PPB) and in vivo brain percentage injected dose (%ID); and Km and specific binding in vivo (BPND) were investigated. Results showed that HPLC measurements of Pm, %PPB and Km were potentially useful in predicting in vivo performance and hence allow evaluation and ranking of compound libraries for the selection of lead radiotracer candidates at early stages of radiotracer discovery. The HPLC tool developed provides information on in vivo non-specific binding and binding potential that is not possible using conventional screening methods. Another important finding reported in this thesis is that Log P should not be relied on as a predictor of brain entry. The HPLC tool developed, together with competition binding assays, was used to characterise a newly synthesised library of compounds for imaging of the translocator protein (TSPO) in brain using SPECT. Results showed that compound LS 1 was the most likely to succeed within the library investigated, but the high %PPB observed for LS 1 suggested novel compounds with improved %PPB were needed. Thus, a novel library of compounds for imaging of TSPO in brain using SPECT is currently been developed for future testing using the HPLC tool developed here and competition binding assays. Pre-clinical research: radiotracers for imaging the noradrenaline transporter (NAT) in brain using SPECT. In this thesis, NKJ64, a novel iodinated analogue of reboxetine, was successfully radiolabelled via electrophilic iododestannylation and evaluated as a potential SPECT radiotracer for imaging the NAT in brain using rodents and non-human primates. Biological evaluation of the novel radiotracer, 123/125I-NKJ64, in rodents included: in vitro ligand binding assays; in vitro and ex vivo autoradiography; in vivo biodistribution studies and ex vivo pharmacological blocking studies. In rats, 123/125I-NKJ64 displayed saturable binding with nanomolar affinity for the NAT in cortical homogenates, regional distribution consistent with the known density of NAT in the rodent brain and high maximum brain uptake of around 2.93 % of the injected dose. The specific: non-specific ratio (locus coeruleus:caudate putamen) of 123I-NKJ64 uptake was 2.8 at 30 minutes post intravenous injection and prior administration of reboxetine significantly reduced the accumulation of 123I-NKJ64 in the locus coeruleus (> 50% reduction). Data obtained using rodents indicated that further evaluation of 123I-NKJ64 in non-human primates was needed to determine its utility as a SPECT radiotracer for imaging of NAT in brain. Consequently, in vivo kinetic modelling studies using SPECT imaging with 123I-NKJ64 and two baboons were carried out to determine 123I-NKJ64 brain binding kinetics, brain distribution and plasma metabolism in non-human primates. Even though a high brain uptake of around 3.0% of the injected dose was determined, the high non-specific binding observed throughout the brain, a low binding potential (BPND<2) in NAT rich regions and a brain distribution that was inconsistent with the known NAT distribution in non-human primate brain precludes the translation of 123I-NKJ64 into humans. Another NAT radiotracer, 123I-INER, developed by Tamagnan and colleagues at Yale University and Institute for Degenerative Disorders, New Haven, USA, was also investigated as part of this thesis. Kinetic modelling analysis of 123I-INER in baboon brain was investigated for different models, namely invasive and reference tissue models. Bolus plus constant infusion experiments with displacement at equilibrium using six different doses of atomoxetine and four different doses of reboxetine were carried out in several baboons to obtain occupancy measurements as a function of injected dose (mg/kg) for the two NAT selective drugs. Results showed that reference tissue models were able to determine BPND values of 123I-INER in different brain regions. In addition the volume of distribution could be determined by dividing concentration in tissue by the concentration in venous blood at 3 hours post-injection. After administration of atomoxetine or reboxetine, dose-dependent occupancy was observed in brain regions known to contain high densities of NATs. Results supported the translation of 123I-INER into humans studies, despite the slow kinetics determined over the imaging period. Pharmacokinetic properties of 123I-INER described in this thesis may be used to simplify future data acquisition and image processing. Conclusion In conclusion, this thesis reported: (1) the development of novel radiotracers for brain imaging, namely NAT and TSPO; and (2) the development of a new methodology for aiding lead molecule identification at early stages of radiotracer discovery (i.e. prior to radiolabelling). In addition, an overview of radiotracer discovery and development process is provided in a single document, with a focus on brain radiotracers.