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Title: Iodoreboxetine : the development of a novel SPECT brain imaging tracer for the noradrenaline transporter
Author: Crawford, Andrew Raymond
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2013
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Abstract:
The noradrenaline system is extensively innervated throughout the brain, implicated in the aetiology of a wide range of psychiatric conditions, and the pharmacological modulation of the noradrenaline system has had a positive influence upon the alleviation of symptoms for those suffering psychiatric or neurological disease. Clinical imaging of the brain with technologies such as Single Photon Emission Computed Tomography (SPECT) can provide clinicians and researchers with valuable information for elucidating disease aetiology, monitoring patient condition, and also confirming the mechanism of action for drug development through occupancy studies. However, to date, there is no SPECT brain imaging tracer for the noradrenaline transporter in regular clinical use. This thesis is a body of work to develop a novel SPECT brain imaging tracer for the noradrenaline transporter with compounds that are iodinated forms of reboxetine, the selective noradrenergic reuptake inhibitor and clinically used antidepressant. From the compounds synthesised and tested, the one which demonstrated the best pharmacological affinity and selectivity for the noradrenaline transporter was NKJ-64. This same compound also displayed many of the HPLC-derived properties predicting in vivo molecular behaviour that are associated with successful brain imaging tracers. Therefore, NKJ-64 was recommended for radiolabelling and further study. PCP MODEL OF THE METABOLIC HYPOFRONTALITY OBSERVED IN SCHIZOPHRENIA: The metabolic hypofunction observed in the prefrontal cortex of schizophrenic patients is mimicked in a rat model via repeated, low-dose phencyclidine administration. The neural mechanisms underlying this hypofrontality are unclear. Whilst dopaminergic transmission is classically associated with aberrant activity in schizophrenia, modulation of noradrenergic neurotransmission is correlated with the alleviation of negative symptoms. Furthermore, the noradrenaline transporter rather than the dopamine transporter functions as the dominant dopaminergic reuptake mechanism in the prefrontal cortex. It was hypothesised that repeated phencyclidine administration in the rat would induce a down-regulation of the NAT. Ligands labelled with [3H] were used for autoradiographic imaging of the noradrenaline, dopamine, and serotonin transporters in phencyclidine-treated and control groups, and [35S]-labelled oligonucleotide probes specific for the mRNA of each transporter were used for in situ hybridisation. The binding densities of the noradrenaline and dopamine transporters were unaltered in the model, however significant selective reductions of serotonin transporter binding sites were measured. The densities of mRNA for all three monoamine transporters were unaltered, so the unaltered densities of noradrenaline and dopamine transporter binding sites and the changes in serotonin transporter binding densities were not the result of altered gene expression. Although the noradrenaline transporter was not directly affected in this model of one particular aspect of schizophrenia and the model is not ideal for demonstrating the capability of a SPECT tracer for the NAT, this does not imply that the noradrenaline transporter is uninvolved in the disease aetiology of schizophrenia or of diminished importance to future schizophrenia studies. The aforementioned correlation of noradrenaline transporter modulation to the alleviation of negative symptoms emphasises the importance of acquiring the clinical ability to assess the density and/or occupancy of the noradrenaline transporter by developing a useful SPECT brain imaging tracer for this site. PHARMACOLOGICAL CHARACTERISATION OF POTENTIAL SPECT BRAIN IMAGING TRACERS FOR THE NORADRENALINE TRANSPORTER: In vivo imaging of the noradrenaline transporter was previously limited to peripheral tracers such as metaiodobenzylguanidine (MIBG), which does not cross the blood-brain barrier, and neuroligands such as radiolabelled desiprimine, which demonstrated problematically high nonspecific binding. Recent efforts in the literature have focused upon modified reboxetine analogues, and some progress was made with the S,S-isomer of iodophenoxy-ring reboxetine, referred to as both INER and IPBM in publication (Tamagnan et al. 2007; Kanegawa et al. 2006). Synthesised iodophenyl-ring compounds in S,S-, R,R-, S,R-, and R,S-isomers were evaluated for their affinity for the NAT. The R,S-isomer demonstrated a very similar level of binding to the S,S-isomer and so this was explored in an iodophenoxy isomer to determine if improvement upon the characteristics of lead iodinated reboxetine compounds in the literature was possible. The R,S-isomer iodophenoxy reboxetine analogues were synthesised with the iodine in the ortho, meta, and para positions of the phenoxy ring and assigned the designations NKJ-64, NKJ-67, and NKJ-68, respectively. NKJ-64 has strong affinity and selectivity for the noradrenaline transporter, with a KD of 8.4 ± 1.7 nM, a 6-fold selectivity for the noradrenaline transporter over the serotonin transporter, and a 63-fold selectivity for the noradrenaline transporter over the dopamine transporter. From the compounds synthesised and tested, NKJ-64 has the most suitable pharmacology to be developed further as a SPECT brain imaging tracer for the noradrenaline transporter and has an affinity in the same order of magnitude as iodinated compounds in the literature, such as the aforementioned INER/IPBM. USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) TO PREDICT IN VIVO CHARACTERISTICS OF TRACER CANDIDATES: The development of any candidate tracer compound must include an evaluation of its in vivo properties. To streamline this process and select only the most viable compounds for further testing, predictors of in vivo molecular characteristics are used to conserve research effort. Measures such as lipophilicity, phospholipophilicity, and plasma protein binding, used to estimate the potential for blood-brain barrier penetration, nonspecific binding, and the likelihood of serum availability would be very time-intensive to determine via traditional bench-top methodologies. High-performance liquid chromatography (HPLC) is a quick, precise, and reliable technique that is a rapid and high-throughput automatable process with exceptional precision that does not rely on the bench-top proficiency of an individual experimenter. The optimisation of gradient elution protocols and formulae to interpret sample retention times on commercially available columns into reproducible standardised units has allowed for the efficient evaluation of tracer candidate compounds. The iodinated reboxetine compounds were compared to examples of both successful and failed neuroimaging tracers. Generally, the evaluations of the successful and failed neuroimaging tracers validated the combined methodologies of the HPLC-derived predictors of in vivo molecular behaviour. In applying these methodologies to the synthesised iodoreboxetine compounds, NKJ-64 displayed many of the HPLC-determined properties that are consistent with successful tracers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.572167  DOI: Not available
Keywords: Q Science (General) ; QH345 Biochemistry ; RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
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