A randomised placebo controlled study to assess the effects of cholinergic treatment on muscarinic receptors in Alzheimer’s disease
- P M Kemp1,
- C Holmes2,
- S Hoffmann3,
- S Wilkinson2,
- M Zivanovic1,
- J Thom1,
- L Bolt3,
- J Fleming3,
- D G Wilkinson2
- 1Department of Nuclear Medicine, Southampton University Hospitals Trust, Southampton, UK
- 2Memory Assessment and Research Centre, Moorgreen Hospital, Southampton
- 3Department of Medical Physics and Bioengineering, Southampton University Hospitals Trust
- Correspondence to: Dr Paul M Kemp, Department of Nuclear Medicine, Southampton University Hospitals Trust, Southampton SO16, 6YD, UK;
- Received 23 December 2002
- Accepted 16 May 2003
- Revised 7 May 2003
Objective:To determine the effects of cholinergic treatment on the muscarinic receptor in patients with Alzheimer’s disease.
Methods:12 patients with mild to moderate Alzheimer’s disease and six controls were studied. The patients underwent ADAS-COG psychometric assessment and SPECT brain imaging with 123I quinuclidinyl benzilate (QNB), to demonstrate the postsynaptic muscarinic M1 receptor, before being randomised in a double blind study to receive either an acetylcholinesterase inhibitor (donepezil) or placebo for four months. Following this, the ADAS-COG and the 123I-QNB receptor scan were repeated. The controls were imaged on one occasion only. All image analyses were undertaken using SPM99.
Results:123I-QNB imaging showed a significant relation between baseline psychometric impairment and deficits on scanning. Both placebo and actively treated groups had reductions in 123I-QNB uptake. Greater reductions in receptor binding were demonstrated in the placebo group than in those receiving active treatment. Intraindividual reproducibility of the 123I-QNB imaging technique appeared highly robust.
Conclusions:The results suggest that 123I-QNB uptake is better preserved in Alzheimer’s disease patients on cholinergic treatment than on placebo. Cholinergic treatment may play a neuroprotective role. Sequential 123I-QNB imaging seems to be a powerful tool in monitoring the response of these receptors to disease modifying treatments.
The deposition of amyloid plaques and tangles within cerebral tissue in patients with Alzheimer’s dementia is associated with disruption of the cholinergic nervous system, causing depletion of the neurotransmitter acetylcholine.1 The administration of acetylcholinesterase inhibitors (AChE-I) appears to provide benefit in approximately 40–50% of patients, albeit temporarily.2 There is also a suggestion from a clinical trial of a neuroprotective effect of AChE-I treatment.3
Iodine-123 quinuclidinyl benzilate (QNB) is a radioactive tracer that can be used to demonstrate in vivo the M1 receptor that is predominantly situated on the postsynaptic muscarinic receptor.4 This pilot study was designed to assess the effects of cholinergic treatment on these postsynaptic receptors using 123I-QNB in vivo imaging in a randomised, double blind, pilot study in patients with Alzheimer’s disease.
Twelve patients who fulfilled the NINCDS-ARDRA criteria5 for probable Alzheimer’s dementia were recruited for this study at the memory clinic, Moorgreen Hospital, Southampton, by a consultant in old age psychiatry. Six healthy controls without a history of head injury or neuropsychiatric illness, and with no apparent cognitive impairment, were also recruited.
The 12 patients with Alzheimer’s disease underwent baseline ADAS-COG (Alzheimer’s disease assessment scale—cognitive behaviour) psychometric assessment and 123I-QNB imaging followed by randomisation in a double blind trial to receive either four months of treatment with an AChE-I (donepezil 5 mg daily increasing to 10 mg at four weeks) or placebo. Following this period, and before discontinuation of the allocated treatment, the psychometric assessment and brain scan were repeated. The six controls were imaged on one occasion only and did not undergo formal psychometric testing.
123I-QNB preparation and imaging
The (R,R) QNB isomer was synthesised by the Department of Radiopharmacy, Glasgow University, and was subsequently labelled locally with 123I using a high performance liquid chromatographic technique.6 Five hours after the intravenous administration of 160 MBq 123I-QNB, the subjects underwent a 30 minute tomographic acquisition on a SMV DST-XL dual head gamma camera. These projections were prefiltered, corrected for decay and attenuation, and reconstructed with a ramp filter. The statistical parametric software package (SPM99) was used for image analysis.7 The reconstructed images were registered to a single photon emission computed tomographic (SPECT) template image set in standardised stereotactic space, smoothed, and normalised to the mean count within the image.8
The following analyses of the SPM maps of the 123I-QNB images were undertaken:
Assessment of associations between the 123I-QNB images and the psychometric assessment scores using linear regression analysis;
Group comparison (t test) of the baseline patient images v the controls;
Group comparison (t test) of the baseline images of the actively treated patient group v the placebo group;
Paired t test of the baseline and four month follow up patient images for those on active treatment;
Paired t test of the baseline and four month follow up patient images for those on placebo;
Group comparison (t test) of the differences between the baseline and four month follow up images for the actively treated group v the placebo group.
Each comparison within SPM99 produced a three dimensional statistical map of voxels showing significantly reduced counts that was displayed in three orthogonal projections. Voxels were included in the map if their uncorrected p value was less than 0.001. The overall statistical significance, referred to as the set level within SPM99, was assessed using a threshold p value of 0.05, either over the whole cortex or over a predefined cortical region.9
The project received approval from the local research ethics committee and the Administration of Radioactive Substances advisory committee; patient consent was obtained in accordance with the declaration of Helsinki.
The median ages of the patient and control groups were 75 years (range 58 to 87) and 70 years (range 65 to 79), respectively; there was no significant difference between medians (Mann-Whitney test, p = 0.20). The ADAS-COG scores of two of the patients in this pilot study were excluded, one because of visual impairment and one because of withdrawal from the trial. The median ADAS-COG scores at baseline of the actively treated and placebo group were 23 (range11 to 34) and 15 (range 9 to 28), respectively; there was no significant difference between medians (Mann-Whitney test, p = 0.27).
All 12 patients successfully underwent the baseline brain imaging and were randomised to receive either a cholinesterase inhibitor or placebo. Unfortunately one patient appeared intolerant of the allocated treatment. For this patient the code was broken and the treatment was found to be the active drug; the patient was therefore removed from the trial. The 11 remaining patients successfully completed the four months of active treatment or placebo, followed by repeat neuroimaging.
The six controls underwent neuroimaging on one occasion only. Jack-knifing the QNB control studies (a procedure whereby each of the six subjects is removed in turn and compared with the remaining five subjects) within SPM99 showed that one of these control subjects had significant abnormalities. A subsequent rCBF SPECT cerebral perfusion study demonstrated significant parieto-temporal hypoperfusion suggestive of early Alzheimer’s disease. Consequently this control was withdrawn from the study and will be kept under medical review.
A significant association was noted between the initial ADAS-COG and the baseline 123I-QNB images such that greater impairment on psychometric testing was associated with reduced uptake of 123I-QNB (p<0.05). This finding was based on the 10 patients who completed brain imaging and had complete ADAS-COG assessment.
No significant group differences (placebo v controls) were noted on the changes in neuropsychometry, nor were any significant associations found between the sequential changes on psychometric assessment and brain imaging in this pilot study.
SPM99 analysis did not show any significant group differences between the baseline scans of the 12 patients and the five subjects in the control group.
A group comparison of the baseline 123I-QNB studies of the six patients on active treatment with the five patients randomised to placebo did not show any significant differences.
The abnormalities demonstrated on imaging for an individual patient showed very similar patterns between baseline and follow up studies for each patient, indicating that the 123I-QNB imaging technique is highly reproducible; however, there was considerable variability in the patterns of abnormalities between patients.
A paired t tests of the patients on active drug showed a reduction in tracer uptake in their four month follow up scan as compared with their baseline scan (311 voxels showed a significant decrease, p = 0.028; fig 1). Similarly, in the placebo group there was a reduction in the corresponding scans (745 voxels showed a decrease, p = 0.016; fig 2). For both these comparisons, up to 300 voxels would have been expected by chance.
A group comparison within SPM99 of the differences of the paired scans in the actively treated group (follow up study minus baseline) with the differences of the paired scans in the placebo group showed greater differences in the placebo group. These changes were centred on the left parieto-temporal region. By restricting the analysis to the posterior cortices (mild to moderate Alzheimer’s disease predominantly affects parieto-temporal grey matter), a significant cluster could be identified in the region of the precuneus in the left medial parietal lobe (p = 0.02).
In vivo imaging of the cerebral postsynaptic muscarinic receptor has been successfully undertaken using 123I-QNB,4,10 which appears to target the M1 muscarinic receptor preferentially11; these receptors are predominantly located postsynaptically. Excellent correlations have been shown in healthy controls between the distributions of 123I-QNB uptake on in vivo imaging and muscarinic receptor densities in vitro.4
Reduced M1 receptor uptake in patients with Alzheimer’s disease has been observed by others,4,10 predominantly in the parieto-temporal cortices. Although this is at variance with several histopathological findings of normal receptor density,12–15 the most likely explanation is that, although the receptors may be structurally present at necropsy, they are non-functioning. A possible mechanism is uncoupling between the M1 receptor and the G protein related signal cascade complex.16
In this pilot study we showed a significant relation between impaired ADAS-COG scores and deficits on 123I-QNB images at baseline. This finding, and the lack of association between changes on psychometric scores and imaging, needs to be interpreted with caution given the small numbers.
Our inability to show significant group differences between the baseline images of the patients and the controls is most likely to be a reflection of several factors including the small number of subjects, the relatively mild disease severity, and—most probably the major factor—the wide interindividual variability of abnormal patient patterns in mild disease states.
In contrast to the considerable interindividual variability between the patterns of abnormalities on the patient scans, all patients showed similar patterns of abnormalities on their individual pair of scans (baseline and follow up), indicating creditable intraindividual reproducibility. Repeat imaging of the controls was not considered appropriate on radiation grounds. However, we believe that the changes in a patient in a four month period would be minimal. This excellent intraindividual reproducibility should enable sequential studies of high power with a relatively modest number of subjects.
Group comparisons of the paired scans using SPM99 showed a significantly greater reduction in the QNB uptake in patients taking placebo than in those on active treatment. Interestingly, the localisation of a predominant cluster in the left precuneus is consistent with recent findings of early involvement of this medial-parietal region in mild Alzheimer’s disease.17,18
It is unlikely that the potentially increased levels of endogenous acetylcholine in the actively treated group would affect the ligand binding. (RR)-QNB has a higher affinity at M1 muscarinic receptors than agonists such as acetylcholine.11,19,20 An in vivo animal study using the positron emission tomography (PET) ligand 18F-FP-TZTP to target the M1 and M2 muscarinic receptors showed only a non-significant reduction in cortical ligand binding when acetylcholine concentrations were increased by the administration of the acetylcholinesterase inhibitor physostigmine.21 In our study, any displacement of the QNB ligand by raised endogenous levels of acetylcholine in the actively treated group would have underestimated the significant differences noted in receptor binding between the actively treated and placebo groups.
The results of this pilot study show that patients on cholinergic treatment have better preservation of M1 receptor binding to 123I-QNB than those receiving placebo. This suggests a possible neuroprotective role for cholinergic treatment in Alzheimer’s disease.
We would like to express our gratitude to the Welcome Clinical Research Facility, Southampton, for the support throughout this project, to Dr M A Piggott, MRC/University Joint Centre Development for Clinical Brain Ageing, Newcastle Upon Tyne, for the advice on aspects of neuropharmacology, to Mrs L Bolton for her administrative and secretarial contribution, and to Messrs J Williams and J Langford for their imaging assistance. The study was funded by grants from PPP Healthcare Medical Trust (AMRC), and HOPE (Wessex Medical Trust), Southampton. PMK was awarded a Research and Development Fellowship from Southampton University Hospitals Trust.
Competing interests: Pfizer-Eisai have awarded a project grant to DW and CH, involved them in multicentre trials, and contributed to their expenses for conference presentations.