Jonathan Owens

Nicotinic Acetylcholine Receptor Distribution in Alzheimer's Disease, Dementia with Lewy Bodies, Parkinson's Disease, and Vascular Dementia: In Vitro Binding Study Using 5-[125I]-A-85380

Nicotinic acetylcholine receptors (nAChRs) have been implicated in a number of neurological disorders. 5-Iodo-3-[2(S)-2-azetidinylmethoxy]pyridine (5-I-A-85380) is a novel nAChR marker, binding predominantly to the α4β2 subtype. This in vitro autoradiography study describes the distribution of 5-[125I]-A-85380 binding in post-mortem brain tissue from normal elderly individuals and from cases with age-associated dementias of both neurodegenerative and vascular types. The binding distribution of 5-[125I]-A-85380 in normal brain tissue was found to be consistent with the reported distribution of other high-affinity nicotinic ligands. In addition to high thalamic and moderate striatal and temporal cortex density, moderate 5-[125I]-A-85380 binding was also seen in white matter tracts in cingulate, occipital, and temporal areas, indicating the presence of nAChRs along nerve fiber tracts, which has not been reported in other high-affinity nicotinic agonist distribution studies. In Parkinsons disease (PD), loss of striatal 5-[125I]-A-85380 binding closely parallels the loss of nigrostriatal dopaminergic markers previously observed. In dementia with Lewy bodies (DLB) reduced striatal 5-[125I]-A-85380 binding density, comparable to that in PD, may be a marker of early degeneration in nigrostriatal inputs, while in Alzheimers disease (AD) reduced striatal 5-[125I]-A-85380 binding could be related to reduced cortical inputs. The reductions of nAChRs seen in AD, DLB, and PD were not apparent in vascular dementia (VaD). In conclusion, 5-I-A-85380 is clearly a useful ligand for both in vitro and in vivo single photon emission tomography human studies investigating disease symptoms and progression, response to acetylcholinesterase-inhibiting drugs and in differentiating primary degenerative dementia from VaD.

Muscarinic receptors in basal ganglia in dementia with Lewy bodies, Parkinson's disease and Alzheimer's disease.

Derivatives of the muscarinic antagonist 3-quinuclidinyl-4-iodobenzilate (QNB), particularly [123I]-(R,R)-I-QNB, are currently being assessed as in vivo ligands to monitor muscarinic receptors in Alzheimers disease (AD) and dementia with Lewy bodies (DLB), relating changes to disease symptoms and to treatment response with cholinergic medication. To assist in the evaluation of in vivo binding, muscarinic receptor density in post-mortem human brain was measured by autoradiography with [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB and compared to M1 ([3H]pirenzepine) and M2 and M4 ([3H]AF-DX 384) receptor binding. Binding was calculated in tissue containing striatum, globus pallidus (GPe), claustrum, and cingulate and insula cortex, in cases of AD, DLB, Parkinsons disease (PD) and normal elderly controls. Pirenzepine, AF-DX 384 and (R,S)-I-QNB binding in the striatum correlated positively with increased Alzheimer-type pathology, and AF-DX 384 and (R,R)-I-QNB cortical binding correlated positively with increased Lewy body (LB) pathology; however, striatal pirenzepine binding correlated negatively with cortical LB pathology. M1 receptors were significantly reduced in striatum in DLB compared to AD, PD, and controls and there was a significant correlation between M1 and dopamine D2 receptor densities. [3H]AF-DX 384 binding was higher in the striatum and GPe in AD. Binding of [125I]-(R,R)-I-QNB, which may reflect increased muscarinic M4 receptors, was higher in cortex and claustrum in DLB and AD. [125I]-(R,S)-I-QNB binding was higher in the GPe in AD. Low M1 and D2 receptors in DLB imply altered regulation of the striatal projection neurons which express these receptors. Low density of striatal M1 receptors may relate to the extent of movement disorder in DLB, and to a reduced risk of parkinsonism with acetylcholinesterase inhibition.

Comparative distribution of binding of the muscarinic receptor ligands pirenzepine, AF-DX 384, (R,R)-I-QNB and (R,S)-I-QNB to human brain

Quinuclidinyl benzilate (QNB) and its derivatives are being developed to investigate muscarinic receptor changes in vivo in Alzheimers disease and dementia with Lewy bodies. This is the first study of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB binding in vitro in human brain. We have compared the in vitro binding of the muscarinic ligands [3H]pirenzepine and [3H]AF-DX 384, which have selectivity for the M1 and M2/M4 receptor subtypes, respectively, to the binding of [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB. This will provide a guide to the interpretation of in vivo SPET images generated with [123I]-(R,R)-I-QNB and [123I]-(R,S)-I-QNB. Binding was investigated in striatum, globus pallidus, thalamus and cerebellum, and cingulate, insula, temporal and occipital cortical areas, which show different proportions of muscarinic receptor subtypes, in post-mortem brain from normal individuals. M1 receptors are of high density in cortex and striatum and are relatively low in the thalamus and cerebellum, while M4 receptors are mainly expressed in the striatum, and M2 receptors are most evident in the cerebellum and thalamus. [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB density distribution patterns were consistent with binding to both M1 and M4 receptors, with [125I]-(R,R)-I-QNB additionally binding to a non-cholinergic site not displaceable by atropine. This distribution can be exploited by in vivo imaging, developing ligands for both SPET and PET, to reveal muscarinic receptor changes in Alzheimers disease and dementia with Lewy bodies during the disease process and following cholinergic therapy.

Deficits in iodine-labelled 3-quinuclidinyl benzilate binding in relation to cerebral blood flow in patients with Alzheimer's disease.

A loss of acetylcholine is one of the most consistent neurochemical findings in Alzheimers disease (AD) post-mortem, but the debate concerning receptor abnormalities is unresolved. The aim of this investigation was to measure the density of acetylcholine muscarinic receptors in AD patients at various stages in the disease (N = 8) by synthesising a radio-iodinated version of quinuclidinyl benzilate QNB, a potent muscarinic antagonist. Deficits were identified by comparison with a control data set obtained from four elderly volunteers and then compared to the deficit in total functional activity in the same brain regions measured using the cerebral perfusion tracer technetium-99m hexamethylpropylene amine oxime. Iodine-123 (R, R)quinuclidinyl benzilate (QNB) was synthesised using a CuI assisted nucleophilic aromatic exchange reaction. 160 MBq of the radioligand (specific activity 400 Ci/mmol: dose 90 ng/kg) was administered to each subject. Diagnosis of AD was made using the CAMDEX and DSMIIIR criteria with a physical examination, full blood screen, CT and chest X-ray. All subjects were scanned at 21 h post injection on an SME810 emission tomograph. 123I(R, R)QNB activity in the controls was found to be consistent with the known distribution of muscarinic receptors with no activity in the cerebellum and low activity in the thalamus. In the AD patients deficits in 123I-QNB binding which exceeded the corresponding total functional regional perfusion deficit were not found in six of the patiens and were observed only in the two most severely affected patients, both of whom were untestable on the cognitive battery. This adds weight to the evidence that a major reduction in postsynaptic receptor density takes place only at a very late stage of AD.

An efficient targeted radiotherapy/gene therapy strategy utilising human telomerase promoters and radioastatine and harnessing radiation-mediated bystander effects

Targeted radiotherapy achieves malignant cell‐specific concentration of radiation dosage by tumour‐affinic molecules conjugated to radioactive atoms. Combining gene therapy with targeted radiotherapy is attractive because the associated cross‐fire irradiation of the latter induces biological bystander effects upon neighbouring cells overcoming low gene transfer efficiency.

In vivo SPECT imaging of muscarinic acetylcholine receptors using (R,R) 123I-QNB in dementia with Lewy bodies and Parkinson’s disease dementia

INTRODUCTION Alterations in cholinergic function have been reported to be associated with dementia. The aim of this study was to investigate differences in the distribution of muscarinic acetylcholine receptors (mAChRs) using (R,R) 123I-iodo-quinuclidinyl-benzilate (QNB) and single photon emission computed tomography (SPECT) in dementia with Lewy bodies (DLB), Parkinsons disease dementia (PDD) and age-matched controls. 123I-QNB binding was also compared to the corresponding cerebral perfusion changes in the same subjects. METHODS 63 subjects (24 controls, 14 DLB, 25 PDD) underwent 123I-QNB and perfusion 99mTc-exametazine SPECT scanning. Image analysis, using statistical parametric mapping (SPM99), involved spatial normalisation of each image to a customised template, followed by smoothing and intensity normalisation of each image to its corresponding mean whole brain uptake. Group effects and correlations were assessed using two sample t tests and linear regression respectively. RESULTS Relative to controls, significant elevation of 123I-QNB binding was apparent in the right occipital lobe in DLB and right and left occipital lobes in PDD (height threshold p<or=0.001 uncorrected). PDD also showed significant loss in uptake in frontal regions and temporal lobes bilaterally that was not present in DLB. These patterns appeared to be independent of any corresponding rCBF changes. CONCLUSION Significant elevation of mAChRs in the occipital lobe was associated with DLB and PDD. This may relate to the visual disturbances that are prevalent in these disorders. Further studies are required in order to establish the role of mAChRs in visual function.

123Iodo-MK-801: A spect agent for imaging the pattern and extent of glutamate (NMDA) receptor activation in Alzheimer's disease

Glutamate, and the NMDA glutamate receptor, may be involved in Alzheimers Disease (AD). Reductions in NMDA receptors are found in AD, possibly contributing to memory deficits. However the NMDA receptor is involved in excitotoxicity, which may play a role in cell death and the production of neurofibrillary tangles in AD; although with lower levels of glutamate than occur in cerebral ischaemia. Therefore reductions in the NMDA receptor may worsen memory deficit in AD, but increased stimulation of the receptor may contribute to the progress of the disease. MK-801 has been used to image excessive glutamate activation following ischaemia in rats. However, it is unclear how effective MK-801 is in conditions with lower levels of glutamate release. This study attempts to gain insight into the utility of the tracer in these conditions, exploring glutamatergic mechanisms in AD. It describes the retention and elimination of 123iodo-MK-801 in five AD and five control subjects, comparing this to regional cerebral blood flow (rCBF). The initial uptake of 123I-MK-801 is dominated by delivery of the ligand. However, despite significant reductions in rCBF in the AD patients, there is no significant difference in the uptake of 123I-MK-801. This suggests increased retention of 123I-MK-801 in the AD patients. In addition the washout of 123I-MK-801 was less in the AD patients, again suggesting increased retention, although this only reached significance in one region. Theses data hint at possible increases in NMDA activation in AD but ultimately 123I-MK-801 does not provide a sufficiently accurate measurement to demonstrate this conclusively. Further NMDA ligands are now at a late stage of development and may provide more conclusive answers to the role of glutamate in AD.

First SPET images of glutamate (NMDA) receptor activation in vivo in cerebral ischaemia

This report describes the initial clinical assessment of (+)-3-[123I]Iodo-MK-801 and its potential to provide single photon emission tomographic (SPET) images in vivo of NMDA receptor activation during cerebral ischaemia. Multiple SPET images were obtained in the 120 min after the administration of 150 MBq of (+)-3-[123I]Iodo-MK-801 to five patients with cerebral ischaemia (due to cerebral haemorrhages) and to five normal volunteers. In normal subjects, (+)-3-[123I]Iodo-MK-801 has a rapid uptake into the brain. The tracer has a high non-specific retention in the central nervous system due to its lipophilicity, which was made evident by the retention of tracer in the cerebellum and white matter (brain areas with few NMDA receptors). In all patients with cerebral haemorrhages, the initial uptake of (+)-3-[123I]Iodo-MK-801 into the ipsilateral hemisphere was markedly reduced, consistent with a reduced level of cerebral blood flow. In two of five patients, relatively increased tracer retention at later time points (60–120 min after tracer administration) could be seen in cortical areas adjacent to the site of the haemorrhage, consistent with activated NMDA receptors. In three of the patients, no relatively enhanced tracer retention could be identified. Using (+)-3-[123I]Iodo-MK-801, it may be possible to image excessive glutamate (NMDA) receptor activation during an ischaemic episode in living human patients. The utility of (+)-3-[123I]Iodo-MK-801 as a SPET ligand for assessing modest alterations in NMDA receptor activity may ultimately be limited by its lipophilicity and consequent high non-specific binding.

Enhanced tumour uptake and in vitro radiotoxicity of no-carrier-added [131i] metaiodobenzylguanidine: Implications for the targeted radiotherapy of neuroblastoma

In vitro and in vivo neuroblastoma models were used to determine whether improvements in tumour targeting in vivo and therapeutic efficacy in vitro could result from the use of no-carrier-added (n.c.a.) [131I]MIBG. Results were compared with use of the conventional therapy MIBG preparation (ex. [131I]MIBG) of lower specific activity which is produced by iodide exchange reaction. The efficacy of n.c.a. [131I]MIBG was compared with that of [131I]MIBG over a range of specific activities by the assessment of neuroblastoma spheroid growth delay. Whereas n.c.a. [131I]MIBG at a radioactivity concentration of 2 MBq/ml prevented the regrowth of 84% of spheroids, toxicity was significantly reduced by the addition of non-radiolabelled MIBG to the incubation medium. The time-dependent biodistribution of n.c.a. [131I]MIBG in nude mice bearing human neuroblastoma xenografts was compared with that of the conventional therapy radiopharmaceutical. The n.c.a. agent gave improved tumour uptake but also significantly greater accumulation in normal tissues known to accumulate MIBG such as heart, adrenal and skin. However, uptake and retention in the blood was unaltered. For all tissues examined, the 3-day calculations were undertaken to predict organ to tumour dose ratios which would result in human neuroblastoma patients with each of the [131I]MIBG preparations. These results suggest that significant therapeutic gain may be achieved by the use of n.c.a. [131I]MIBG as a treatment agent in neuroblastoma. neuroblastoma.

Acetylcholine muscarinic receptors and response to anti-cholinesterase therapy in patients with Alzheimer's disease.

Abstract. An acetylcholine deficit remains the most consistent neurotransmitter abnormality found in Alzheimers disease and various therapeutic agents have been targeted at this. In this study we investigated the action of Donepezil, a cholinesterase inhibitor that has few side-effects. In particular we set out to investigate whether muscarinic acetylcholine receptor (mAChR) availability influences the response to this therapy. We used the novel single-photon emission tomography (SPET) tracer (R,R)[123I]I-quinuclidinyl benzilate (R,R[123I]I-QNB), which has high affinity for the M1 subtype of mAChR. Regional cerebral perfusion was also assessed using technetium-99m hexamethylpropylene amine oxime. We investigated 20 patients on Donepezil treatment and ten age-matched controls. The results showed a reduction in (R,R)[123I]I-QNB binding in the caudal anterior cingulate in patients compared with controls and relatively high binding in the putamen and rostral anterior cingulate, suggesting a relative sparing of mAChR in these regions. The main finding of the study was that mAChR availability as assessed by (R,R)[123I]I-QNB binding did not distinguish responders from non-responders. Interestingly, we found that the extent of cognitive improvement showed no positive correlation with (R,R)[123I]I-QNB binding in any brain region but was inversely related to binding in the insular cortex. This suggests that, within the advised cognitive performance band for use of Donepezil, response is greater in those patients with evidence of a more marked cholinergic deficit. A larger study should investigate this.