The aims of the present thesis were to explore novel candidate radiotracers intended for imaging of dementia by means of molecular imaging techniques, and to evaluate the value of existing radiotracers that are currently being used, or could potentially be used for imaging of dementia. In this chapter, the studies that were performed to accomplish these aims, are summarized.
Chapter 2 describes the synthesis of a series of iodinated TZTP-derivatives as potential radiotracers for imaging of muscarinic M 2 receptors. Such tracers would allow visualization and quantification of the presynaptic muscarinic receptor system by means of SPECT or PET and may be of value to monitor disease progression and effectiveness of experimental therapeutic interventions in neurpsychiatric diseases characterized by a central cholinergic deficit, e.g., Alzheimer's disease (AD). In the reported study, derivatives of 5-[(4-alkylthio)-1,2,5-thiadiazol-3-yl]-1,2,3,6-tetrahydro-1-methylpyridines (TZTPs), originally described by Sauerberg  and proposed as potential tracers for muscarinic receptors [2, 3], were synthesized and tested for affinity to cloned human muscarinic receptor subtypes by in-vitro competitive binding assays. Several of these compounds showed a high affinity for this receptor, within the nanomolar range. The most promising of the novel potential tracers, the iodinated 5-(E)-iodopentenylthio-TZTP, showed a high affinity to M 2 receptors ( 4.9 nM) and favorable selectivity ratios over M 1 and M 3 receptors, was radioiodinated and subsequently subjected to in-vivo biodistribution and blocking experiments in rats. Also, the metabolism of this derivative was studied by TLC. Distribution of the potential tracer in the rat brain occurred in a M 2 -like pattern but blocking effects of the potent muscarinic antagonist scopolamine were only detected until 15 min after injection of the potential tracer, and thus specific in-vivo binding could not be demonstrated after that time point. The value of the new tracer proved to be limited by high non-specific uptake of the original tracer due to high lipophilicity and rapid metabolism of the compound.
Chapter 3 discusses another series of in-vitro competition studies, using newly synthesized compounds based on 6ß-acetoxynortropane, a tropane alkaloid that was reported earlier to have high affinity and selectivity for the muscarinic M 2 receptor subtype as compared to the other muscarinic receptor subtypes . Due to the nature of the molecular tropane skeleton, many derivatives, such as [ 123 I]/[ 18 F]FP-CIT or [ 123 I]ß-CIT, have proven to be useful as radiotracers for receptor imaging in the living human brain. The challenge of this experiment was to create a derivative of 6ß-acetoxynortropane suitable for radioiodination, while preserving the affinity for the M 2 receptor. Moreover, we also attempted to optimize lipophilicity as compared to the TZTP-derivatives that are described in chapter 2, and to improve metabolic stability while maintaining the size of the molecule as small as possible. Four analogues of the molecule were synthesized, each containing an iodophenyl or phenyl moiety that could potentially be radioiodinated for use as radiotracers for imaging of the muscarinic M 2 receptor subtype in neurodegenerative or neuropsychiatric diseases. As in chapter 2, the affinity of these derivates for muscarinic receptor subtypes was assessed in-vitro by competitive binding assays on muscarinic receptors. Unfortunately, substitution of an iodophenyl or phenyl moiety on the original compound, severely reduced both the affinity and selectivity of the derivatives for the muscarinic M 2 receptor subtype and therefore it was concluded that the synthesized analogues were not suitable for use in human SPECT imaging.
In chapter 4, an alternative method for biodistribution experiments in small animals, which is used in chapters 5 and 8 of this thesis, is validated. The conventional dissection technique that is described in chapter 2 may not be adequate to study the biodistribution of radiotracers in small or complex brain structures in small laboratory animals such as rats or mice. Autoradiography using X-ray film allows much more precise measurements, but does not show a linear response to the amount of radioactivity that is exposed to the film . Moreover, X-ray film autoradiography requires long exposure times and therefore it is not suitable for imaging of short-living radioisotopes such as 123 I . Storage phosphor imaging plates are used extensively in digital X-ray diagnostics and also increasingly for autoradiography in radiotracer biodistribution studies, although the response of these plates to short-living radioisotopes is largely unknown. In chapter 4, the properties of the imaging plates are compared directly for 11 short-living radioisotopes ( 18 F, 32 P, 67 Ga, 89 Sr, 99m Tc, 90 Y, 111 In, 123 I, 125 I, 131 I and 201 Tl). Linearity, sensitivity, efficiency and resolution were measured for all isotopes. A linear response over a wide range of applied radioactivity was demonstrated for all tested isotopes. The plates showed the highest efficiency and sensitivity for strong ß - -emitters and the ß + -emitter 18 F, whereas lower efficiency and sensitivity was detected for ?-emitters. For the ?-emitters, only low energy ?-radiation seemed to interact substantially with the plate. The resolution of the images that could be obtained proved to be worse for high energy ß-emitters whereas the best resolutions were obtained with low energy ?-emitters such as 123 I (0.32 mm full width at half maximum; FWHM). Also an ex-vivo study is presented in chapter 4, in which the biodistribution of the dopamine transporter tracer [ 123 I]FP-CIT was studied in the rat brain, with and without blocking of the receptor by pre-administration of methylphenidate, using either the conventional dissection technique or the storage phosphor imaging technique. The results of both studies proved to be comparable but the results that were obtained from the imaging plates showed less variability. It is concluded from chapter 4 that, although the properties in the imaging plates varies considerably for various radioisotopes, the storage phosphor imaging technique is a very attractive alternative to conventional dissection studies when using short-living radioisotopes such as 123 I.Chapter 5 describes the appliance of storage phosphor imaging in an experiment that assessed the ex-vivo binding in the rat brain of a recently proposed radioiodinated tracer for SPECT imaging of activated NMDA receptors, [ 123 I]CNS-1261 . Excessive activation of NMDA receptors, the most abundant receptor of the glutamatergic neurotransmitter system, is believed to contribute to neuronal death in neurodegenerative diseases such as AD or Lewy body disorders . Inhibition of NMDA receptor activation by substances such as memantine , may have neuroprotective effects and (semi)quantitative imaging of the activated system may be useful to select patients for such inhibition therapies. Chapter 5 first reports on a time course dissection biodistribution experiment in rats, which determined the time-point at 2 h after injection as optimal for studying of the tracer distribution in the brain. Second, an experiment is described in which the distribution in the rat brain was studied using storage phosphor imaging in control animals versus animals that were pretreated with either D-serine alone or D-Serine+MK801. D-serine is a substance that co-activates the NMDA receptor and pre-administration should therefore hypothetically lead to a higher binding of [ 123 I]CNS-1261, but this, however, could not be demonstrated. Vice versa, MK801 blocks the binding of the radiotracer to its binding site and proved to decrease the uptake of the radiotracer in brain areas that express relatively high densities of NMDA receptors. Although this decrease was significant as compared to control rats, strongly suggesting specific binding of the tracer, the observed reduction of binding was small and this is also very suggestive of a high degree of non-specific uptake of the tracer. Thus, our study has shown that [ 123 I]CNS-1261 binding is influenced by NMDA receptor availability. However, high non-specific uptake may limit quantification and small changes in receptor availability are unlikely to be detected and therefore its use in human SPECT imaging of activated NMDA receptors may be limited.
Another radiotracer that has proven to be of value for SPECT imaging in dementia is [ 123 I]FP-CIT. This dopaminergic transporter tracer, originally developed for the detection of degeneration of the nigrostriatal projection in Parkinson's disease (PD), was registered recently to differentiate AD from dementia with Lewy bodies (DLB) by means of brain SPECT imaging. DLB is the second most common form of dementia after AD, and is often clinically misinterpreted as AD. In contrast with AD, DLB is characterized by pronounced dopaminergic cell loss , and can thus be differentiated from AD by [ 123 I]FP-CIT SPECT.
In chapter 6, the effects of AChEIs on the binding of [ 123 I]FP-CIT to dopamine transporters are reported. Apart from dopaminergic deficits, there is also a cholinergic deficit in DLB and cognition of such patients is improved by early AChEI therapy . Since many patients that are referred for [ 123 I]FP-CIT brain SPECTs already use medication of this type, and since cholinergic medication may influence the dopamine transporter availability, it is of considerable interest to know the influences of AChEIs on the [ 123 I]FP-CIT binding in the brain. In chapter 6, data is presented from a dissection experiment in which rats were treated with the AChEIs rivastigmine or donepezil or the potent dopamine transporter antagonist methylphenidate. The animals were treated either once intravenously or once orally shortly before injection of the tracer, or during 14 consecutive days (subchronic group) before injection of the tracer. As expected, the methylphenidate group showed a significant decrease in [ 123 I]FP-CIT binding in the rat brain at 2 h after injection of the tracer, after both intravenous and oral pre-administration shortly before injection of the tracer, but not after subchronic administration of the tracer. Neither acute nor subchronic pre-administration of both AChEIs influenced the binding of the tracer in the rat brain. It is concluded that in rats, administration of the tested AChEIs does not lead to an important alteration of the dopamine transporter availability in the brain. Therefore, it is unlikely that AChEIs will induce large effects on the interpretation of [ 123 I]FP-CIT SPECT scans in humans.
Chapter 7 also deals with the effects of psychopharmaceuticals on the binding of a neuroreceptor radiotracer. Similar to the experiment that is discussed in chapter 6, the experiment that is described in this chapter addresses the influences of repeated administration of neuroleptics on the binding of [ 123 I]Iododexetimide in the rat brain. [ 123 I]Iododexetimide was previously developed and evaluated as a non-subtype selective muscarinic receptor SPECT tracer [12, 13], and would therefore perform less well as compared to muscarinic receptor subtype selective radiotracers, if these would be available. However, since a marked overall decrease of muscarinic receptor density has been shown in AD, DLB and PDD [14, 15], this tracer may be of interest to study the integrity of the total muscarinic system in such patients (see chapter 9 for a human study using this tracer). Unfortunately, many of these patients use neuroleptics, which primarily target the dopaminergic system but which may indirectly influence the cholinergic system, and therefore it is important to know the effects of these types of medication on the binding of [ 123 I]Iododexetimide in the brain. In chapter 7, an animal experiment is presented that evaluates potential influences of repeated administration during 14 consecutive days of the atypical neuroleptics olanzapine and risperidone on the binding of [ 123 I]Iododexetimide in the rat brain. Tracer uptake in muscarinic receptor-rich brain areas (i.e. cortical areas and striatum) and in brain areas expressing relatively low levels of muscarinic receptors (i.e. hypothalamus) was compared to data obtained from control rats. Since we were particularly interested in subchronic effects, radiotracer uptake was evaluated after one day of washout of the neuroleptics. No influences on tracer binding were detected after treatment with olanzapine. In the risperidone group, only an unexpected increase in tracer binding was found in the hypothalamus as compared to the control group. Since in-vivo studies in humans can only accurately assess muscarinic binding in muscarinic-rich brains areas, the data suggest no large non-acute effects of olanzapine and risperidone on muscarinic receptor imaging in humans, when using [ 123 I]Iododexetimide.
Chapter 8 further reports on the association between the dopaminergic and the cholinergic system. In this chapter, data are presented from an animal experiment in which rats were given a unilateral selective dopaminergic brain lesion, and the effects of this lesion on the distribution of [ 123 I]Iododexetimide in the rat brain are revealed. The dysfunctional cholinergic neurotransmitter system in PD, PDD and DLB is thought to contribute to the cognitive deficits in these diseases . Besides degeneration of the cholinergic system, dopaminergic disruption which also occurs in these diseases, may directly influence the function of the cholinergic system. In-vivo imaging of the cholinergic system in such patients may be of value to monitor central cholinergic disturbances and to select cases in which treatment with cholinesterase inhibitors could be beneficial. To study the effects of a selective dopaminergic lesion on the [ 123 I]Iododexetimide binding, a group of rats was given an unilateral 6-hydroxydopamine lesion, and the damage to the dopaminergic system was confirmed by small animal SPECT imaging using [ 123 I]FP-CIT. Also, the effects of the lesion on the brain perfusion were measured using SPECT imaging and a significantly lower perfusion was detected in the striatum on the side of the lesion but not in the ipsilateral cerebral cortex. Then, the effects of the lesion on the binding of [ 123 I]Iododexetimide in a series of brain structures was measured using the storage phosphor imaging technique that is described in detail in chapter 4. The phosphor imaging data revealed a consistent and statistically significant lower tracer binding in all examined neocortical areas on the ipsilateral side of the lesion as compared to the contralateral side. In the hippocampus and subcortical areas such as the striatum, such asymmetry was not detected. The data that are presented in this chapter suggest that evaluation of the muscarinic receptor availability in the dopamine depleted brain, using [ 123 I]Iododexetimide is feasible, and also that a 6-hydroxydopamine lesion induces a decrease of neocortical muscarinic receptor availability. This may be due to downregulation of postsynaptic muscarinic M 1 receptors, or direct competition of acetylcholine as a result of hyperactivation of the cortical cholinergic system in response to the disruption of the dopaminergic pathway.
Chapter 9 presents a human [ 123 I]Iododexetimide [12, 13] SPECT study that was performed in PD and PDD patients. Since [ 123 I]Iododexetimide SPECT may help to select cognitively compromised patients that could benefit from AChEIs or to monitor disease progression, this study was conducted to investigate the differences in muscarinic receptor availability in the brain of PDD versus PD patients, which was measured in-vivo. The SPECT studies were performed in 13 subjects (6 PD, 7 PDD) and the obtained brain SPECTs were fitted to a standard brain template, normalized to non-specific tracer uptake in white matter and analyzed using statistical parametric mapping (SPM99). No increases in tracer binding were detected in the brain of PDD versus PD patients. However, significant decreases in tracer binding in the PDD group as compared to the PD group were detected in both the left and right temporal cortex, left hippocampus, and the posterior cingulate cortex. The data that are presented in chapter 9 demonstrate decreased muscarinic receptor binding by [ 123 I]Iododexetimide in PDD versus PD in brain areas that are involved in memory function. The findings suggest that reduced muscarinic receptor binding in these brain areas could be used as a marker for PDD. Additionally, in-vivo [ 123 I]Iododexetimide SPECT-imaging might be of value for the selection of PDD patients that are likely to respond to AChEIs.
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General discussion, future perspectives and conclusions
Due to demographic ageing of the population, a substantial increase in the incidence of dementia is expected in the near future. The progressive course of the disease increasingly interferes with daily tasks in such patients, which puts a serious burden on care-givers and public healthcare. Dementia is therefore prone to develop into a major medical, social and economic problem  . In the past decades, the etiology of dementia has increasingly gained attention in the scientific literature, and this has led to a better understanding of the neuropathological mechanisms that are involved in the various types of dementia.
Today, dementia is still a clinical diagnosis, but the available diagnostic criteria for the various types of dementia are frequently limited in terms of sensitivity and specificity. Complementary use of biomarkers may assist in the diagnostic process. A biomarker is being defined as a measurable indicator of disease activity , is based on a relevant pathophysiological aspect of the disease, is ideally measurable in all patients and is able to measure the effects of treatment . Data obtained from medical imaging studies are often suitable for use as a biomarker, and might be of interest for either clinical or scientific purposes. In this respect, biomarkers may contribute to the early diagnosis of dementia, the differentiation between the various types of dementia or the prediction of treatment response. However, biomarkers may also be used to monitor disease activity or to evaluate the effectiveness of experimental treatments in clinical trials. At present, the main purpose of medical imaging studies in patients suffering from dementia, is to exclude potentially treatable causes of the condition. Additionally, in-vivo anatomical measurements of brain structure volumes are performed routinely and imaging studies that evaluate brain metabolism or perfusion are also carried out. Such imaging studies contribute to the clinical diagnostic process to some extent. Molecular imaging has the potential to selectively target a particular aspect of the pathophysiological mechanism of the disease that underlies the dementia. Therefore, data obtained from molecular imaging studies may be very suitable for use as biomarkers. In the past decades, much effort has been put in the development of in-vivo imaging procedures that could differentiate between the various types of dementia, or reflect specific neuropathological mechanisms of the underlying disease, using nuclear medicine techniques. Although many (candidate) radiotracers have been developed and evaluated for this purpose, only few of these tracers are currently being used in the daily clinical care. Apart from compounds that image brain metabolism or brain perfusion, the only radiotracers that are used routinely to differentiate between types of dementia are dopamine transporter tracers. There are two reasons for this. The fisrst reason is that most candidate tracers that have been developed have proven to be unsuitable for use as radiotracers in patient care, usually due to low signal-to-noise ratios or other unfavorable in-vivo characteristics. However, tracers have been developed that would, after decent evaluation in clinical trials, be appropriate for use in patients that suffer from dementia. This applies to, for instance, recently developed tracers that target nicotinic receptors, muscarinic receptors, or ß-amyloid containing plaques, although the exact value of such tracers as biomarkers for each of these categories is still under debate. The second reason seems to be at least of equal importance. Although every patient benefits from an accurate diagnosis, essential for optimizing all aspects of care, no effective therapy for dementia is available to date, and this may be a reason for the apparent lack of interest of clinicians for these new imaging strategies. As mentioned above, one exception is the SPECT or PET evaluation of the dopaminergic transporter status in patients that suffer from dementia with Lewy bodies (DLB), which is being performed to differentiate this type of dementia from Alzheimer's disease (AD) . In the case of DLB, an accurate diagnosis is important, since these patients may have very serious adverse effects, i.e. acute Parkinsonian crisis, when treated with neuroleptics. It is conceivable that this consideration stimulates clinicians to request a dopamine transporter scan.
However, due to the present scientific interest for dementia and the increasing knowledge of its pathophysiological determinants, it is very likely that better therapies will be developed for the disease in the future. Many pharmaceuticals that may alter the course of dementia, are under investigation at present. In this respect, drugs are being developed that prevent the fibrillisation of ß-amyloid or the formation of plaques, such as anti-ß-amyloid vaccines. Other drugs that target the same pathophysiological mechanisms include ß- and ?-secretase inhibitors, statines and selective muscarinic M 1 receptor agonists. Another promising group of drugs are the neuroprotective agents, such as anti-oxidants, actrocyte modulating substances, NMDA receptor antagonists, anti-inflammatory drugs, AMPA receptor modulators, nicotinic receptor agonists and drugs that target the synthesis of the tau protein .
Prevention of dementia may become a very important issue once effective therapies have been developed. When pharmaceuticals are available that are able to slow down the progression of dementia, or can reverse specific pathophysiologic events of the disease (e.g. anti-Aß-vaccine), or even medication that simply decrease the symptoms of the disease (such as more effective acetyl cholinesterase inhibitors; AChEIs), early detection of the disease becomes much more important. This will inevitably increase the clinical need for proper biomarkers, and these could be provided by molecular imaging. Therefore, it is of crucial importance to expand the molecular imaging toolbox with novel, well characterized radiotracers that are potentially suitable for imaging of specific neuropathological aspects of dementia. However, in scientific research, suitable radiotracers that could act as biomarkers or even surrogate markers (biomarkers that can be used as endpoint in clinical trials ) for dementia are already highly requested. Thus, the synthesis of novel candidate radiotracers and in-vitro and in-vivo characterization of such compounds, and even the evaluation of new methods for neuropharmacologic research, with respect to imaging procedures for dementia, is important.
Most of the experiments that are reported in this thesis implicate the cholinergic neurotransmitter system. Disturbances of this system, which are present in dementia, are thought to be an important contributor to the cognitive dysfunction in these patients [6, 7] and this neurotransmitter system is therefore a potential target for novel radiotracers intended for imaging of dementia. Ever since the formulation of the cholinergic hypothesis for AD [8, 9], much effort has been put in the development of tracers that selectively target a part of this system. The holy grail of such radiotracers could be a selective radiotracer for the muscarinic M 2 receptor subtype , since multiple previous studies have shown that the density of this particular receptor is decreased in patients that suffer from AD. The most promising radiotracer that is being developed in this field is [ 18 F]FP-TZTP , which at least displays selectivity for the M 2 receptor in-vivo . To date, this PET tracer has not been introduced in clinical medicine, but it has been evaluated in normal elderly controls and subjects that genetically predisposed for AD. In this thesis, a study is reported in which we synthesized analogues of this tracer as potential SPECT tracers, and one of these candidate tracers showed favorable in-vitro characteristics, although the selectivity for the M 2 receptor was somewhat limited. Unfortunately, the candidate tracer proved to be metabolized very rapidly in-vivo and showed a high non-specific uptake in the brain, probably due to its high lipophilicity and thus, the tracer was considered unsuitable for use in humans. From the literature it can be told that such adverse in-vivo characteristics are observed frequently in the development of new radiotracers that are intended for use in brain imaging. Other attempts to synthesize a M 2 selective radiotracer, were based on the earlier described, very M 2 receptor selective compound 6ß-acetoxynortropane , but the newly synthesized analogues failed in the in-vitro experiments, not only due to severely decreased affinity for muscarinic receptors as compared to the lead compound, but also due to a loss in selectivity for the M 2 receptor. Both iodinated analogues of TZTP and 6ß-acetoxynortropane, or similar analogues that could potentially be iodinated, resulted in compounds with inferior in-vitro or in-vivo binding characteristics than the original molecules. The synthesis of a potent, iodinated, selective and metabolically stable tracer for the M 2 receptor proved to be difficult. Based on the knowledge of molecules that bind selectively to M 2 receptors, it seems conceivable that the first appropriate tracer that will be developed for the M 2 receptor, will be a 18 F or 11 C labeled PET-tracer. Reasons for this include the more favorable in-vivo characteristics of such tracers after radiolabeling, since no iodine atom is necessary in the molecule, but also because the imaging technique is gaining popularity fast and becomes increasingly available.
Whether a selective M 2 receptor has to be pursued is also debatable. Although a decline in M 2 receptors is characteristic for AD, there is also proof for a decline of receptors of the nicotinergic neurotransmitter system, and these may precede the decline of M 2 receptors, so targeting the nicotinic system may be more appropriate with respect to the early diagnosis of AD. Recent advances in the development of nicotinic receptor radiotracers [14, 15] implicate that targeting of this neurotransmitter system may be an attractive alternative for the exploration of disturbances of the cholinergic system in AD. However, receptor selective muscarinic radiotracers may gain new attention due to the role of the muscarinic M 1 receptor in the APP cleavage process  and tracers for the M 1 receptor may therefore be useful for the prediction of response to M 1 agonists. Imaging of the M 1 receptor may also be of value for the evaluation of other neuropsychiatric diseases. A subgroup of patients that suffer from schizophrenia that shows a vast decline of cortical muscarinic M 1 receptor density , has recently been identified and this offers opportunities for both treatment and molecular imaging.
All in-vivo experiments that are reported in this thesis use [ 123 I] labeled radiotracers. Experiments in rats in which the biodistribution was investigated in small or complex brain structures, necessitated a more sensitive experimental method than the classical dissection technique, in which brain structures are dissected and counted for radioactivity separately. Conventional autoradiography is a very precise method, but could not be used due to the short half-life of 123 I , but a similar method, storage phosphor imaging , proved to perform very good in this respect. Although this technique is used frequently in biodistribution experiments, the characteristics of the phosphor plates for short-living radioisotopes had never been evaluated systematically for a larger series of isotopes. The study that is reported in this thesis shows that the phosphor imaging technique is suitable for use with tracers that are labeled with short-living radioisotopes, such as 123 I, and that this method is valuable for pharmacologic experiments in the brains of small animals using radioactive tracers. Although the maximal resolution is much lower than that of conventional autoradiography, the storage phosphor imaging technique has some important advantages. Advantages are e.g. the short exposure periods that are necessary to obtain images and the high capacity of the plates which prevents overexposure. For imaging of microscopic structures, the conventional autoradiography films remain to be the method of choice. As compared to dissection techniques, the storage phosphor imaging technique is more precise in measuring radioactivity in small and complex brain structures such as the hippocampus. However, the method is rather labor-intensive and when a fast screening in a large group of animals is to be performed, the dissection technique is more attractive. As compared to small animal SPECT procedures, the resolution of the phosphor imaging technique is superior, but whether this is also the case when compared with micro-PET, has to be established.
In this thesis, the phosphor imaging technique was used for an experiment in which the in-vivo characteristics of [ 123 I]CNS-1261, a candidate radiotracer that targets activated NMDA receptor [20, 21], were explored. Activation of these receptors is a non-specific characteristic of neurodegenerative diseases and this mechanism is therefore potentially of interest for imaging and quantification of disease activity in patients with dementia . In this study, we showed that the binding of the tracer could partially be influenced, indicating specific binding of the tracer to the NMDA receptor, but also that a high nonspecific uptake occurred in the brain, which may be the result of fast in-vivo metabolism of this tracer. Indeed, this has also been reported by another group , and this renders the value of this tracer for use in humans as limited.
The value of dopamine transporter tracers, such as [ 123 I]FP-CIT has been emphasized above, and this particular tracer is now used clinically for the differentiation between AD and DLB. Similar to patients that suffer from Parkinson's disease (PD), DLB patients show a marked decrease of dopamine transporter density in the brain , which discriminates this type of dementia from AD. Knowledge about the effects of drugs on the outcomes of neuroimaging procedures is crucial, since patients may need to discontinue the medication before such scans are being performed. In this thesis, an animal experiment is presented in which the effects of AChEIs, which are used frequently by both AD and DLB patients, on the binding of [ 123 I]FP-CIT are evaluated. Although interactions between the dopaminergic and the cholinergic are well known, no effects of administration of the AChEIs donepezil and rivastigmine, on the binding of the tracer to the presynaptic dopaminergic nerve terminals were revealed. Effects of other AChEIs such as galantamine are also expected to have no effects on the [ 123 I]FP-CIT binding in the brain. These results are in line with a recently performed retrospective study , in which data from human studies were evaluated, and which also showed no effects of concurrent use of AChEIs on obtained images that reflect the dopamine transporter status in DLB patients. However, the retrospective character of this particular study does not provide the ultimate proof, and a radomized trial should ideally be performed to prove that AChEIs does not interfere with [ 123 I[FP-CIT imaging. Until then, there is no need for discontinuation of AChEIs before a [ 123 I]FP-CIT scan is being performed.
Potentially interfering effects of medication on the binding of [ 123 I[Iododexetimide were also evaluated. This tracer binds non-selectively to all muscarinic receptor subtypes en has favorable in-vivo characteristics [25, 26]. Since the density of muscarinic M 1 receptors in the human brain is much higher than the density of M 2 receptors, this tracers reflects predominantly the muscarinic M 1 receptor status, when used in imaging studies. The tracer may therefore be of clinical value for the SPECT imaging of the postsynaptic muscarinic neurotransmitter system, and may be useful for the prediction of therapy response to AChEIs in DLB, although an earlier report using a similar radiotracer, R,R [ 123 I]QNB, failed to distinguish responders from non-responders within a group of AD patients . Non-subtype selective muscarinic receptor tracers may also prove to be useful for the group of schizophrenia patients, which exhibited a profound loss of cortical M 1 receptors, according to the study by Scarr et al. . Patients with either dementia or schizophrenia frequently use neuroleptics, and this has been the motivation to study the effects of subchronic administration of neuroleptics (olanzapine and risperidone) on the binding of [ 123 I]Iododexetimide in the rat brain. The experiment revealed no effects of neuroleptics on the binding of the tracer in brain areas that are known to express a high density of M 1 receptors. Thus, there seems to be no necessity to discontinue the use of neuroleptics before a [ 123 I]Iododexetimide scan is being performed. Human data on potential influences of neuroleptics on the binding of [ 123 I]Iododexetimide are not available yet, and this issue should be further studied before this tracer will be used in patient groups that use this type of medication.
However, alteration of the dopaminergic system has been shown in this thesis, to influence the binding of [ 123 I]Iododexetimide in the brains of rats. To detect these small influences, we used the sensitive storage phosphor imaging technique. Unilateral damage to the dopaminergic neurotransmitter system due to an induced brain lesion, revealed a decrease of tracer binding in ipsilateral neocortical brain areas of lesioned rats. This is presumably the result of hyperactivation of the muscarinic neurotransmitter system [28, 29], which is thought to occur due to the release from the inhibitory actions of dopaminergic system that had been damaged. It is hypothesized that the decreased binding of [ 123 I]Iododexetimide is the result from either a downregulation of postsynaptic M 1 receptors, or competition of acetylcholine on the binding of the tracer to the receptor. One potential limitation of this study is the fact that the animals served as their own control group, since binding of [ 123 I]Iododexetimide was compared for the lesioned versus the unlesioned hemisphere. There are indications for minor effects of the unilaterally performed dopaminergic lesion on the contralateral dopaminergic system , and this can not be excluded in this study. In further studies using this animal model, a sham operated control group should therefore be included.
The effects of a decreased dopaminergic inhibition on the binding of [ 123 I]Iododexetimide are likely to be small and presumably not detectable with SPECT, which is a less sensitive imaging technique as compared to phosphor imaging. For imaging of neurodegenerative diseases that are accompanied by a marked decrease in muscarinic receptor density, hyperactivity of the cholinergic system will not be an important issue.
In the human SPECT study, where [ 123 I]Iododexetimide was used to detect differences in tracer binding between PDD and PD patients, potential effects of the release of the muscarinic system from the inhibition by the dopaminergic system are most likely non-existent. De Hoehn and Yahr scores of the patients of both groups were similar and showed no significant difference. The demonstrated decline of [ 123 I]Iododexetimide binding in brain areas that are important in memory function, in PDD versus PD patients, may therefore be the result of degenerative changes in the muscarinic neurotransmitter system, as would be expected in PDD. This study suggests that non-subtype selective muscarinic receptor tracers may be of value for the detection of deficiencies in the cholinergic system, although it can not be excluded that differences in perfusion or regional atrophy account for the observed decreased tracer binding . In future studies, measurements on atrophy by MRI or CT techniques, or measurements on cerebral perfusion would be a valuable addition, although the acquisition of multiple scans may be difficult in this particular population. From a scientific point of view, it would be interesting to determine whether prediction of treatment response is feasible by performing [ 123 I]Iododexetimide brain SPECTs in PDD patients. However, it has become clear that the observed small decreases in tracer binding do not allow prediction of treatment response in individual patients, although this verdict would be strengthened after data is also obtained from an age-matched control group. The homogenous distribution of muscarinic receptor subtypes in the human brain will complicate the visual interpretation of [ 123 I]Iododexetimide SPECTs in diseases that only show small alterations of muscarinic receptor densities. In the subgroup of schizophrenia patients from the postmortem study by Scarr et al. , a 74% decrease of [ 3 H]pirenzipine binding to muscarinic M 1 receptors was detected, and in such patients, [ 123 I]Iododexetimide SPECT should be perfectly adequate for the visual determination of the muscarinic receptor status in individual patients.
The aims of this thesis were to explore novel candidate radiotracers intended for imaging of dementia by means of molecular imaging techniques, and to evaluate the value of existing radiotracers that are currently being used, or could potentially be used for imaging of dementia.
In conclusion, it can be stated that attempts to synthesize a selective muscarinic M 2 receptor radiotracer have led to disappointing results. All candidate radiotracers have failed either in-vitro or in-vivo. The experimental radiotracer intended for imaging of activated NMDA receptors also proved to be unsuitable for use in human imaging, due to unfavorable in-vivo characteristics of the compound. Existing neuroreceptor tracers seem to have more potential for use in clinical diagnostics. A good example is the dopamine transporter tracer [ 123 I]FP-CIT, which is now used clinically for the differentiation between DLB and AD. In rats, we did not find evidence for interfering effects on the binding of this tracer to its target by AChEIs. Since similar results have been reported in humans, there is no reason to discontinue this type of medication before an [ 123 I]FP-CIT SPECT is being performed. Also no effects of subchronic administration of neuroleptics were detected on the binding of [ 123 I]Iododexetimide in the rat brain, and therefore there seems to be no reason for discontinuation of these pharmaceuticals before perfoming a [ 123 I]Iododexetimide SPECT. However, this should be verified in humans before this tracer is being used clinically. Small effects of dopaminergic deficiency have been shown in rats, but are not likely to influence the interpretation [ 123 I]Iododexetimide SPECTs in patients. A decrease of [ 123 I]Iododexetimide binding in the brains of PDD patients has been shown as compared to PD patients, but is of a small magnitude and probably not visually detectable in individual patients. A non-subtype selective muscarinic receptor radiotracer such as [ 123 I]Iododexetimide is likely to be of value for the evaluation of diseases of the central nervous system, that are accompanied by a larger decrease in muscarinic receptor density.
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