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Parkinson's disease: monitoring early diagnosis and disease progression

Winogrodzka, A.

2007

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https://research.vu.nl/en/publications/7f0acf24-49b0-4c08-bfbd-99411c51ab2f

parkinson’s disease monitoring early diagnosis and disease progression

Ania Winogrodzka

Copyright: ©Hanna Winogrodzka, Amsterdam 2007

ISBN: 9789086591480

Typesetting: Michał Sławiński

VRIJE UNIVERSITEIT

Parkinson’s disease

monitoring early diagnosis and disease progression

Academisch Proefschrift

ter verkrijging van de graad Doctor aande Vrije Universiteit Amsterdam,op gezag van de rector magnificus

prof.dr. L.M. Bouter,in het openbaar te verdedigen

ten overstaan van de promotiecommissievan de faculteit der Geneeskunde

op donderdag 8 november 2007 om 13.45 uurin het auditorium van de universiteit,

De Boelelaan 1105

door

Hanna Winogrodzka

geboren te Jaslo, Polen

promotor: prof.dr. E.Ch. Wolters copromotor: prof.dr. R.C. Wagenaar

Contents

7 Chapter 1

General introduction

1.1 General background of Parkinson’s disease1.2 Identification of early Parkinson’s disease1.3 Outline of the thesis

17 Chapter 2SPECT imaging of the presynaptic dopaminergic system and early clinical features of Parkinson’s disease (nonlinear dynamics perspective)2.1 Rigidity decreases resting tremor intensity in Parkinson’s disease:

an [123I]ß-CIT SPECT study in early, non-medicated patients2.2 Rigidity and bradykinesia reduce interlimb coordination in

parkinsonian gait

43 Chapter 3Leads for the development of neuroprotective treatment in Parkinson’s disease and brain imaging methods for estimating treatment efficacy

59 Chapter 4SPECT imaging of the presynaptic dopaminergic system and progression of dopaminergic degeneration in Parkinson’s disease

4.1 Dopamine transporter SPECT is a useful method to monitor dopaminergic degeneration in early-stage Parkinson’s disease

4.2 Disease-related and drug-induced changes in dopamine transporter expression might undermine the reliability of imaging studies of disease progression in Parkinson’s disease

85 Chapter 5General discussion and future perspectives

93 Chapter 6Summary

97 Chapter 7Samenvatting

101 Chapter 8

Reference list

Publications

Dankwoord

CHAPTER 1


Chapter 1

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1.1 PARKINSON’S DISEASE: GENERAL BACKGROUND

1.1.1 Epidemiology

Parkinson’s disease (PD) is the most common movement disorder and second most common neurodegenerative disorder after Alzheimer’s disease. It affects about 1% of adults older than 60 years1. The estimated prevalence for PD is 1.6%, increasing with age2. People of all races and ethnic origins can be affected3 and the incidence is some-what higher among men than among women4. As the result of its chronic and pro-gressive course, which often leads to severe disability, PD has a major socio-economic impact on society. Despite many advances in understanding and treatment, much is still unknown about this progressive neurologic disorder.

1.1.2 Pathology and pathogenesis

The classical pathological finding in PD is slow but progressive loss of neurons in the substantia nigra pars compacta and ventral tegmental area accompanied by the presence of intraneuronal Lewy bodies in the remaining neurons. Lewy body is not pathognomonic of PD, for it has been described in other disorders such as dementia5. According to novel neuropathological findings of Braak and colleagues6, nigral neurons are not the only cells affected in PD. Multiple extranigral neuronal systems such as norepinephrine neurons in the locus coeruleus, cholinergic neurons in the nucleus basalis of Meynert, serotonin neurons in the dorsal raphe and neurons of the dorsal motor nucleus of the vagus become damaged, with the most early neuropathological changes starting in the medulla oblon-gata/pontine tegmentum and olfactory bulb/anterior olfactory nucleus. The biochemical consequence of this process is a profound decrease in striatal dopamine concentration combined with varying degree of deterioration of the cholinergic, serotonergic, and nor-adrenergic system. In this thesis we will further focus on the dopaminergic system.

In the majority of patients, the cause of the disease remains a mystery but the cur-rent insight is that a combination of ageing, background genetic susceptibility and certain environmental triggers may be responsible for the development of the illness. Although over the last few years, several genes for rare, monogenically inherited forms of PD have been mapped, no genetic factors have been clearly associated with increased risk for spo-radic PD7. Up till now no clear environmental causes of PD have been identified3.

1.1.3 Clinical features

Although some believe that Leonardo da Vinci in the 15th century possibly had already described the abnormalities that appear in patients with PD8, it was an English phy-sician, James Parkinson who in 1817 gave a more detailed description of its clinical

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picture and natural history in his Essay on the Shaking Palsy9. He was the first to estab-lish the signs and symptoms of tremor, bradykinesia and gait and postural disturbances into a recognised clinical entity. Parkinson’s description however, was based on only six individuals (three of whom observed on the streets of London) and was incomplete making, for example, no note of rigidity. Four decades later Jean-Martin Charcot added rigidity to Parkinson’s clinical description and attached the name Parkinson’s disease to the syndrome.

According to the current clinical definition, PD is a syndrome characterised by the presence of core features: tremor, rigidity, bradykinesia and postural instability. The combination of those features is also called parkinsonism. Most patients have a majority of these cardinal symptoms at some point during their illness, allowing the clinical diag-nosis of the disease. The onset of motor symptoms in PD is typically asymmetric. A rest-ing limb tremor with a frequency of 4–6 Hz is found in over 70% of PD patients at pre-sentation. It remains the most characteristic feature of PD, but other forms of tremor can also be seen in otherwise typical patient, such as: orthostatic tremor and action tremor. Rigidity is an increased resistance of a joint to passive movement throughout the whole range of motion and may be experienced by patients as stiffness and pain. Bradykinesia and hypokinesia, meaning slowness and poverty of movements, affect all voluntary and involuntary movements and are very disabling symptoms of early PD. While rigidity and bradykinesia in PD are generally thought to result from degeneration of the dopaminer-gic nigrostriatal pathway and consequent reduction in striatal dopamine, the pathofysi-ology of resting tremor is still unclear. Postural instability, often considered as another cardinal feature of PD, is usually absent in early disease. It refers to gradual development of poor balance leading to falls. Gait abnormalities such as pronounced slowing of gait, shuffling and freezing, are also rarely prominent early in the course of PD.

Besides motor features, various non-motor manifestations of PD (such as auto-nomic, sensory, cognitive and mood impairment, as well as sleep and olfactory dysfunc-tion) may be present, probably reflecting degeneration of non-dopaminergic systems and depending on the extent of this degeneration and the disease progression.

1.1.4 Diagnosis

In spite of recent advances in structural and functional neuroimaging, the diagnosis of PD still remains to be based on clinical grounds. There are no biological or imaging markers for the antemortem diagnosis in PD. The current clinical criteria according to UK Parkinson’s Disease Society Brain Bank include bradykinesia in combination with at least one of the following: rigidity, 4–6Hz resting tremor and postural instability10. Unilateral onset and persistent asymmetry of the symptoms, presence of the rest tremor, progressive nature of the disease, clinical course longer than 10 years as well as sustained improvement of motor symptoms with levodopa are features supporting a diagnosis of PD. A definite diagnosis of PD is based on autopsy.

Chapter 1

10

1.1.5 Differential diagnosis

It is estimated that idiopathic PD accounts for about 75% of all cases of parkinsonism11. Since about 25% of all patients with parkinsonian features have an alternative diagnosis, one should be aware of the list of alternative diseases involving primary parkinsonism (such as genetic parkinsonism, progressive supranuclear palsy, multisystem atrophy, de-mentia with Lewy bodies, corticobasal degeneration), as well as secondary parkinsonism (such as vascular and drug-induced parkinsonism, psychogenic parkinsonism, toxins, infections, structural brain lesions, metabolic disorders and trauma)12.

1.1.6 Natural course and prognosis

The expected natural course of PD is a progressive motor and non-motor decline. It is generally accepted that clinical (motor) signs of PD become evident when about 80% of striatal dopamine concentration and 50% of nigral neurons are lost13. Manifest PD is preceded by an early, premotor phase in which subtle non-specific non-motor symptoms as well as “soft” motor signs can occur. The duration of this premotor stage of PD is still a matter of debate and was in previous studies estimated to vary from a few years to sev-eral decades13,14,15, mostly assuming linear progression of neuronal loss in the early stage. Recent evidence from longitudinal imaging studies however, suggests that the progres-sion of PD might follow an exponential course, slowing down in more advanced stages of the disease and that the preclinical period lasts approximately 6 years with a substan-tial loss of dopaminergic neurons at the time of symptom onset16. This is consistent with the results of clinical longitudinal studies17 as well as with autopsy data13. There is also evidence for variable progression rates between individual PD patients. Patients with early cognitive disturbance, older age at the onset of PD symptoms and lack of tremor at presentation generally tend to have a more rapidly progressive disease18.

1.1.7 Therapy Standard symptomatic therapy

Current therapeutic strategies for motor features of PD are symptomatic and focus pri-marily on reducing the severity of the symptoms by correcting the striatal dopamine de-ficiency with dopamine suppleting and/or substituting medications. Dopamine precur-sor, levodopa is still considered the mainstay of symptomatic medical treatment in PD since its introduction more than 30 years ago. Although providing substantial improve-ment, benefits are relatively short-lived and long-term use of levodopa is associated with significant disabling complications, mainly due to progressive course of the disease and pulsatile character op dopaminergic receptor stimulation. These complications include motor side effects such as mid-dose and/or diphasic dyskinesias and motor response fluctuations (wearing off and on-off phenomenon) as well as neuropsychiatric compli-

11


cations. Dopamine agonists also provide anti-parkinson benefit, although perhaps to lesser extent than levodopa. They are however, rather associated with non-motor side-effects such as hypersexuality and psychosis than with motor side-effects19.

A number of other, non-dopaminergic medications such as amantadine and anti-cholinergics can be used alone or in combination with levodopa and dopamine agonists to provide some symptomatic effects in patients with PD. In order to prolong the dura-tion of levodopa induced dopamine receptor stimulation monoamineoxidase-B (MAO-B) and catechol-O-methyltransferase (COMT) inhibitors have been developed20. In order to achieve better therapeutic effect, recently alternative continuous dermal, intra-cutaneous and duodenal drug administration strategies have been introduced, bypass-ing the oral route21.

Functional neurosurgery for PD is indicated when medical therapy cannot suf-ficiently control symptoms. The currently accepted procedures include either CNS le-sions or high frequency deep brain stimulations and target on the subthalamic nucleus, the ventral intermediate nucleus of the thalamus and globus pallidus internal-segment as well as pedunculopontine nucleus and prelemniscal radiation22,23. Due to non-motor adverse events however, deep brain stimulation of the subthalamic nucleus is less ap-plicable in PD patients suffering from pre-existent or concomitant, whether or not PD related psychiatric symptoms24.

Other treatment approaches are rehabilitation, including physical therapy with external cue programmes25, speech therapy and occupational therapy, as well as general lifestyle modifications.

Neuroprotection

Although the symptomatic dopaminomimetic treatment approach is effective in the early stages of PD, the progressive decline in dopaminergic neurons and the devel-opment of pharmacodynamically and pharmacokinetically induced complications of therapy in advanced PD result in profound functional disability. In addition, the patients can progressively suffer from features that do not respond to dopaminergic therapy. At present, no treatment can influence the progressive course of the disease. The great hope and challenge for effective management of PD is the development of neuroprotective therapy to prevent further neuronal degeneration, thereby slowing down or halting disease progression. Growing insight into mechanisms underlying cell death in PD has provided several candidate drugs/interventions which can be consid-ered as putative neuroprotective treatment to be tested in clinical trials in PD26. Before considering the clinical application of those treatments it is important to realize that at the time of the initial clinical presentation of PD there is already substantial loss of dopaminergic, as well as other central nervous system neurotransmitter systems. Therefore, these treatments will bring most benefit when started before the onset of clinical symptoms. This early treatment strategy requires a biological marker of early/premotor disease. Additionally, to measure the efficacy of neuroprotective interven-

Chapter 1

12

tions in PD patients, it is important to develop reliable endpoints, not confounded by the symptomatic or regulatory effects of study drug itself, which would reliably reflect the impact of neuroprotective interventions on disease progression.

1.2 IDENTIFICATION OF EARLY PD

The presence of a premotor phase in PD gives a unique opportunity for presymptom-atic diagnosis and neuroprotective intervention, provided that we are able to accurately detect the pathological process early in its course. A number of attempts have been made to identify biomarkers of early PD. The early clinical prodromes may consist of subtle motor abnormalities (including changes in handwriting, clumsiness, decreased arm swing when walking, subtle asymmetric hypokinesia, fatigue and abnormalities of visuomotor control) as well as non-motor symptoms (such as mood and personality disturbances, subtle neurocognitive dysfunction, vegetative and sensory signs and sleep disturbances) which may even precede the first motor hallmarks of the disease. Any of these signs and symptoms alone, however, is not specific for PD but taken together they might be suggestive for the developing disease27. Applying tools such as personality questionnaires, neuropsychological tests and visuomotor testing procedures might help to identify persons at risk of developing PD. Olfactory loss is a common non-progres-sive feature in most PD patients, even at the earliest clinical stages of the disease and of-ten preceding the onset of motor symptoms by several years28, 29. Olfactory dysfunction was also found in asymptomatic relatives of PD patients30,31, suggesting that olfactory testing might be a potential tool to indicate preclinical diagnosis. One must be cautious, however, since olfactory dysfunction is not specific for PD and may occur in many other settings such as Alzheimer’s disease and head trauma.

Genetics may provide another excellent assessment of the persons at risk in fami-lies with autosomal dominant inherited disease, but this is not generalizable to sporadic PD, since no definitive genetic abnormality underlying the common sporadic form of PD has yet been discovered.

Another possibility to detect early PD might be offered by morphological and functional neuroimaging. Recently, transcranial ultrasound imaging showed increased signal intensity of the substantia nigra in PD patients compared with normal controls32. Hyperechogenicity of the substantia nigra seems to reflect the iron content in the ni-gral tissue and is thought to be a susceptibility marker for PD. The finding of hyper-echogenicity of the substantia nigra in elderly persons with subtle hypokinesia, but not fulfilling diagnostic clinical criteria for idiopathic PD raised the speculation that this ultrasound measure also may serve as a marker for nigral injury and eventual marker of preclinical PD33. As of yet the sensitivity and specificity of this procedure in detecting early disease in PD needs to be established.

Functional imaging of central noradrenergic, cholinergic en serotonerg systems, especially imaging of presynaptic dopaminergic system might also play an important

13


role in early PD detection as well as in monitoring of disease progression and will be discussed in the subsequent sections of this thesis.

1.2.1 Subtle motor abnormalities

A number of studies proposed different methods for identifying and quantifying early motor abnormalities in incipient PD. The traditional research on locomotion in PD has focused on kinematic, kinetic, and electromyographic changes in the lower extremities and identified typical characteristics such as an overall lower speed, smaller stride length, shuffling steps and reduced flexion and extension ranges of the joints. However, those features have a low sensitivity and specificity for early diagnosis. The methods and tools from the dynamical systems theory of movement coordination may offer a unique oppor-tunity to study the early changes in Parkinsonian gait. Previous research on the dynamics of gait suggested that gradual manipulation of a single control parameter such as walking speed, results in important transitions in arm and leg movements as well as pelvic and thoracic rotation, expressed as relative phase between body segments (order parameter) within the normal human walking mode. The ability to make transitions between coor-dination patterns is an important issue in the evaluation of movement disorders. In PD patients overall reduced adaptations in coordination patterns between arms and legs as well as pelvic and thoracic rotations were found as compared with healthy subjects. The relative phase changes identified differences between PD patients and healthy subjects, while no such differences were observed in the traditional stride parameters, such as stride length and stride frequency. With the progression of the dopaminergic degenera-tion and clinical deficit in more advanced PD the changes in gait patterns occur. It is sug-gested that the flexibility and stability of coordination pattern (relative phase analysis) may serve as a sensitive indicator that could be utilized in early disease detection34,35.

1.2.2 Functional imaging of the presynaptic dopaminergic system

Over the past two decades functional imaging with positron emission tomography (PET) and single photon emission computed tomography (SPECT) has been used to assess do-paminergic function in vivo in PD patients. A number of PET and SPECT tracers are now available. The two most widely used imaging biomarkers of the presynaptic dopaminergic function are 18F-Dopa PET providing a measure of the striatal accumulation and me-tabolism of dopamine, and dopamine transporter (DAT) imaging with PET and SPECT, reflecting the density of dopamine transporters. DAT is a protein localized on the pre-synaptic dopaminergic nerve terminal, which controls synaptic dopamine levels by active re-uptake of dopamine from the synaptic cleft back into the presynaptic nerve ending.

In this thesis we will focus on DAT imaging. Several radioligands are now avail-able for this purpose. Tracers designed for PET imaging are: 11C-CFT, 18F-CFT and 11C-RTI-32, while SPECT tracers include: 123I-β-CIT, 123I-FP-CIT, 123I-altropane, and 99mTc-

Chapter 1

14

TRODAT-1. The most widely used are 123I-β-CIT and 123I-FP-CIT. Numerous clinical imaging studies with these tracers have shown bilateral reductions in DAT ligand up-take in the putamen and caudate in PD patients as compared with controls, providing a sensitive measure for discriminating patients in even early stage of PD from healthy people36. The reduction in DAT is both region specific (putamen>caudate) and asym-metric, consistent with pathologic assessment of DAT loss as well as with clinical pre-sentation37. Cross-sectional evaluations showed good correlation between overall stria-tal DAT radioligand uptake and measures of disease severity, with a progressive decline of DAT binding with increasing locomotor disability 38,39 (Fig.1). DAT SPECT has been also shown to have good test/retest reproducibility in patients and controls40.

Progress in functional brain imaging offers promise for the potential of DAT im-aging in the evaluation of premotor nigrostriatal dysfunction and the detection of sub-clinical PD. Many studies showed that striatal DAT concentration measured by SPECT and PET is reduced by 50% at symptom onset, suggesting that this technique should be able to detect very early nigral dysfunction. There is already evidence suggesting that DAT imaging can identify subjects in very early stages of PD or even in the premotor phase of the disease. In early hemiparkinsonian patients there was a significant bilateral reduction in DAT uptake41,42, also contralateral to the still clinically intact body side. Another example is abnormal reduction in striatal DAT uptake in hyposmic relatives of sporadic PD patients, some of whom later developed clinical parkinsonism43.

Based on the present evidence, it can be concluded that the assessment of dopa-mine terminal function with DAT imaging provides a sensitive marker for the presence of PD and gives the opportunity for detection of premotor dopaminergic dysfunction.

PD is a progressive disorder. Despite development of excellent clinical rating scales there are a number of problems undermining the use of these parameters to assess the progressing disability in PD. The fact that they are subjective and easily influenced by

Figure 1. DAT SPECT images of a healthy volunteer (A), an early PD patient (B). Transverse slices at the level of the striatum.

A B

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symptomatic antiparkinsonian therapy is the most bothersome. Although discontinua-tion of the medication can partly overcome this problem, the wash-out period required to obtain a drug-naïve situation may be as long as several weeks. This underscores the need for objective measures to monitor the loss of dopaminergic function in vivo. Using DAT imaging with PET and SPECT, potential objective biological markers for monitor-ing disease progression in PD might be provided. This is highly relevant from the per-spective of interventions that attempt to slow the rate of dopaminergic degeneration.

1.3 CONCLUSIONS AND OUTLINE OF THE THESIS

To date the only therapies of proven efficacy in the treatment of PD are symptomatic. Nevertheless, patients eventually experience progressive disability because of decreasing quality of drug response and development of new, therapy-nonresponsive symptoms. The growing awareness of the need for better long-term management of PD stimulated the concept of disease-modifying interventions which would alter the course of PD by preventing or retarding the clinical progression. Successful development and implemen-tation of such therapies requires not only understanding of the cause and the etiopatho-genesis of the disease but also the possibility of identification of subjects in a very early or even premotor stage of PD, since disease modification seems most feasible early in the course of the disease. There is also a need to define reliable outcome measures to estimate the extent and the progression of dopaminergic degeneration in the living patient as well as methods which would enable us to adequately confirm neuroprotective effects.

In this thesis we firstly focused on some early clinical (motor) features of PD and examined them from the perspective of dynamical theory of movement coordination. Our observations on the relation between tremor and rigidity as well as the effect of rigidity and bradykinesia on interlimb coordination in early PD patients, both made in the context of early nigrostriatal degeneration measured with β-CIT SPECT, are re-ported in chapters 2.1 and 2.2. Those observations provide more insight in the nature of the clinical signs of PD early in its course, which might be valuable in the early detec-tion of the disease. Secondly, the etiopathogenetic mechanisms of PD are highlighted within the frame of oxidative stress theory and leads for the development of putative neuroprotective strategies are discussed (chapter 3). In search for a tool which could be used to measure the effects of putative neuroprotective interventions we investigated the utility of β-CIT and FP-CIT SPECT in the assessment of the rate of progression of dopaminergic degeneration in early PD patients. We also estimated the sample size nec-essary to demonstrate a significant effect of a putative neuroprotective agent in future trials (chapter 4.1). In chapter 4.2 the reliability of DAT imaging as a surrogate marker for disease progression and neuroprotection is discussed, with a special attention for the evidence of regulatory effects of drugs on DAT radiotracer binding.

In chapters 5 and 6 the results of the findings presented in this thesis are summa-rized and discussed and the directions for future research are outlined.

CHAPTER 2.1

Rigidity decreases resting tremor intensity in Parkinson’s disease:

a [123I]β-CIT SPECT study in early, non-medicated patients

A Winogrodzka, RC Wagenaar, P Bergmans, A Vellinga, J Booij, EA van Royen, REA van Emmerik, JC Stoof, ECh Wolters

Movement Disorders 2001;16:1033–1040

Chapter 2.1

1�

Summary

Tremor is one of the clinical hallmarks of Parkinson’s disease (PD). Although it is ac-cepted that other classical symptoms of PD, such as rigidity and bradykinesia, result from a degeneration of the nigrostriatal system and subsequent reduction in striatal do-pamine, the pathophysiology of resting tremor remains unclear. The majority of recent SPECT and PET studies, using various radioligands demonstrated significant correla-tion between striatal radioligand bindings and the degree of parkinsonian symptoms, such as rigidity and bradykinesia, but not tremor. We investigated the relationship between the degeneration of the nigrostriatal pathway and the appearance of resting tremor, taking into account the possible interference of rigidity with the resting tremor. Thirty early and drug-naive PD patients were examined. Tremor and rigidity of the arms were assessed using UPDRS, and the power of tremor was estimated using spec-tral analysis of tremor peaks. [123I]β-CIT SPECT was used to assess degeneration of the dopaminergic system in PD patients. A comparison between the asymmetry indices showed that in terms of both tremor and rigidity, the most affected arm corresponded significantly with the contralateral striatum, having the largest reduction in radioligand binding. Furthermore, tremor power accounted for a significant part of variance in the contralateral striatum, suggesting a relationship between this PD symptom and the degeneration of the dopaminergic system. Further, the degree of tremor was reduced with increasing rigidity. However, correcting for the influence of rigidity the significant contribution of tremor in the variance in the contralateral striatal [123I]β-CIT bind-ing disappeared. When the confounding influence of rigidity is taken into account, no significant direct relationship between dopaminergic degeneration and the degree of tremor could be found. Other pathophysiological mechanisms should be similarly in-vestigated in order to further understand the origin of resting tremor in PD.

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Rigidity decreases resting tremor intensity in Parkinson’s disease: a [123I]β-CIT SPECT study in early, non-medicated patients

Introduction

Together with rigidity and bradykinesia, tremor is one of the clinical hallmarks of Parkinson’s disease (PD). This involuntary oscillation comprises alternate contractions of antagonistic muscles with a higher amplitude and lower, remarkably stable, frequen-cy (about 4–5 Hz)44 as compared to the physiological tremor of healthy subjects (about 10–20 Hz). PD tremor is usually present at rest but can be quite variable even within one subject. Resting tremor, postural tremor, action tremor and the cogwheel phenom-enon are all forms of tremulous movements in PD45. A number of studies indicate that movements such as walking and finger tapping (with or without external auditive rhythm) can result in the disappearance of both ipsilateral and contralateral resting tremor34,35,46,47. It has also been suggested that maneuvers designed to overcome the thixotropic stabilization of the muscles can reveal tremor which is not clinically appar-ent in PD patients48. Because approximately 30% of PD patients never develop tremor, distinguishing between tremor-dominant and an akinetic-rigid form of the disease has been suggested49.

Although it is accepted that rigidity and bradykinesia in PD result from degene-ration of the dopaminergic nigrostriatal pathway and consequent reduction in striatal dopamine50, the pathophysiology of resting tremor is still unknown45,51. Different re-sponses of the main PD symptoms to dopamine replacement therapy also suggest that, to a certain extent, different neuronal mechanisms are involved in the development of the major symptoms. For example, L-DOPA administration is very effective in alleviat-ing rigidity and bradykinesia (especially in the beginning of treatment), but is less ef-ficient in reducing the resting tremor.

Single Photon Emission Computed Tomography (SPECT) with the radioligand [123I]β-CIT has been used to visualize the density of dopamine transporters, which are located on the terminals of dopaminergic projections in the striatum52. [123I]β-CIT binding in PD patients has previously been shown to be significantly decreased in the caudate and putamen as compared to controls53-55. Furthermore, significant correlation has been reported between the reduction in [123I]β-CIT striatal binding in PD patients and Hoehn and Yahr staging37,41,56. Likewise, results of a number of recent [123I]β-CIT SPECT and 6-L-[18F]fluorodopa Positron Emssion Tomography (PET) studies show sig-nificant correlations between the decrease in the caudate and putaminal ligand binding and the degree of bradykinesia37,57 and rigidity42,58,59. In the above mentioned studies such relationship has not been found between ligand binding with SPECT or PET and the severity of tremor. It has also been suggested that, apart from nigrostriatal lesion, other mechanisms are involved in the emergence of resting tremor60. Although this might be true, some confounders could possibly have interfered with the assessment of the resting tremor, which may have been responsible for the lack of significant cor-relation. For example, interference of rigidity with the severity of tremor has never been investigated.

Chapter 2.1

20

We hypothesize that the limb rigidity in PD patients reduces the severity of rest-ing tremor and thus prohibits a significant relationship between the degeneration of the nigrostriatal pathway and resting tremor. In 30 non-medicated PD patients, rigidity and resting tremor of the arms were assessed by means of the Unified Parkinson’s Disease Rating Scale (UPDRS), and spectral analysis of accelerometer signals was performed to provide a further estimate of the power of tremor. [123I]β-CIT SPECT was used to deter-mine the extent of degeneration of the dopaminergic system.

Material and Methods

Patients

Thirty early and non-medicated PD patients were studied. Inclusion criteria comprised: 1) diagnosis of PD according to the criteria of the UK Parkinson’s Disease Society Brain Bank61; 2) a positive result of the apomorphine challenge test (subcutaneously injected dose of 3 mg), defined as minimally 20% reduction in the UPDRS motor score62; 3) no record of dopaminergic medication; 4) the Mini Mental State Examination score above 23; and 5) sufficient motivation.

The study was approved by the medical ethical committee of the University Hos-pital Vrije Universiteit; all patients provided written informed consent.

Procedure

PD patients were assessed by history, physical examination, tremor registration and [123I]β-CIT SPECT. Tremor and rigidity were scored with the UPDRS, and staged with the Hoehn and Yahr scale. Tremor oscillations were registered with accelerometers dur-ing 60 sec. During this procedure, patients were in a sitting position with the arms loose along the sides of the body, and were instructed not to suppress the tremor in any way. Subsequently, SPECT imaging was performed. [123I]β-CIT was injected intravenously at an approximate dose of 110 MBq after treatment with potassium iodide, in order to block thyroid binding of free radioactive iodide. SPECT image acquisition was per-formed 24 h post-injection63.

AssessmentUPDRS

In order to assess the influence of rigidity on the appearance of resting tremor, UPDRS tremor scores and rigidity scores (range 0–4) of the arm most affected by tremor (ipsi-lateral) were used. Tissingh et al.42 reported a significant correlation between the stria-tal [123I]β-CIT binding and the UPDRS rigidity score, but not UPDRS tremor score. Therefore, the UPDRS tremor scores were compared with the resting tremor measured

21


with accelerometers. For this purpose the assessment was made at the right and left arm separately. In addition, the asymmetry in rigidity (RA) as well as tremor (TA) between left and right body sides were assessed by means of the following indices:

RA=(Rr-Rl) over {(Rr+Rl)/2}

RA=Rr−Rl

RrRl /2

TA=(Tr-Tl) over {(Tr+Tl)/2}

TA=Tr−Tl

TrTl /2

where Rr and Rl represent the UPDRS rigidity scores of the right and left arm, respec-tively, and Tr and Tl the UPDRS tremor scores of the right and left arm, respectively.

Accelerometry

Tremor oscillations were recorded with uniaxial accelerometers (Coulbourn type T-45; sample frequency 104 Hz). The accelerometer signals were amplified through a trans-ducer-coupler (Coulbourn A-s72-25). The accelerometers were attached to the proces-sus styleoideus radii of both arms. Frequency analysis of the spectral components of the acceleration time series was performed. The power spectrum was estimated by a Fast Fourier Transformation algorithm using the Welch method for power estimation, and a Hanning window for smoothing the signals (Matlab inc.64). The relative power of the tremor peak within the power spectrum range 3–9 Hz was established by dividing the power of the tremor peak by the mean power of the total spectrum.

Asymmetry in the tremor power between the left and right body side (PA) was assessed with the following asymmetry-index:

PA=(Pr-Pl) over {(Pr+Pl)/2}

PA=Pr−Pl

PrPl /2

where Pr represents the power of the tremor in the right arm and Pl the power of the tremor in the left arm.

SPECT

The Strichman 810X system was used for SPECT imaging (Strichman Medical Equipment, Medfield, MA). The transaxial resolution of this camera is 7,6 mm full width at half maximum of a line source in air. The energy window was set at 135–190 keV. 123I labeling, acquisition, attenuation correction and reconstruction of images were performed as earlier described42. Data acquisition took place in a 128×128 matrix. The measured concentration of radioactivity was expressed as Strichman Medical Units (SMUs; 1 SMU = 100 Bq/ml). For analysis of striatal

Chapter 2.1

22

[123I]ß-CIT binding, two transversal slices representing the most intense striatal binding were summed. A standard region of interest (ROI) template, constructed according to a stereotactic atlas and including regions for striatum and occipital cortex, was placed bilaterally on the combined image, as described previously65. Estimates of specific striatal binding were made by subtracting occipital counts (non-specific binding) from striatal counts. The ratio of specific to non-specific striatal [123I]β-CIT binding was then calculated. A SPECT asymmetry-index (SA) was introduced in order to calculate right to left asymmetry of [123I]ß -CIT binding to the dopamine transporters in the striatum:

SA=(Sr-Sl) over {(Sr+Sl)/2}

SA=Sr−Sl

SrSl /2

where Sr represents the ligand binding in the right striatum and Sl the ligand binding in the left striatum.

Statistical analysis

Binomial analysis was applied to test the correspondence between the asymmetry indices. Because of skewed distribution of tremor at the right and left side, the re-lationship between UPDRS tremor score and tremor power was measured using Spearman’s rank correlation coefficient. Relationships between other variables were assessed using Pearson correlation coefficients. For assessing the relationship be-tween rigidity and striatal [123I]ß-CIT binding ratio, and between tremor power and [123I]ß-CIT binding ratio, ANOVA was used. The correction for the influence of ri-gidity on resting tremor was performed by computing the tremor-Z-score in each rigidity category (UPDRS 1, 2 and 3) because, due to empty cells, it was not possible to correct for rigidity by adding it as second factor. For further exploring the pos-sible relationship between radioligand binding and tremor power, tremor power was divided into five categories.

For all tests 0.05 was chosen as the level of significance. The Bonferroni correc-tion was used for multiple comparisons (significance indicated by *). All analyses were carried out with SPSS v.7.5 software (SPSS, Chicago, IL).

Results

Mean disease duration of the patients was 2.3 years (SD=1.2), whereas the mean Hoehn and Yahr stage was 1.5 (SD=0.5). Twenty one patients had initial right-sided motor signs of PD and nine patients had left-sided onset (mean age, 52.8 years; range 32–64 years). Clinical characteristics, UPDRS scores for tremor and rigidity, tremor power and SPECT values are presented in table 1.

23


Tremor: accelerometry versus UPDRS

Although no prominent tremor scores were found upon clinical examination (maximal UPDRS tremor score was 2), accelerometry measures showed a very stable (5-6 Hz) resting tremor with high power in 18 patients (see figure 1). The UPDRS tremor scores in those patients varied from 1 to 2 points. In another 11 patients, spectral analyses of the acceler-ometry signals demonstrated resting tremor activity, which was not observed during clini-cal examination (UPDRS score= 0). Historical information from these patients revealed past tremulous manifestations. In one patient tremor was not observed either during clini-cal examination or accelerometry, nor had the patient noticed any tremor in the past. A significant positive correlation was found between UPDRS tremor score and tremor power for the right arm (Rs=0.44; p=0.014*), whereas the correlation between UPDRS tremor score and tremor power for the left arm approached significance (Rs=0.36; p=0.05).

Table 1. Clinical, demographic, tremor power, and specific to non-specific striatal [123I]β-CIT binding ra-tios of 30 patients with Parkinson’s disease*

Mean Std. Deviation Minimum MaximumAge 52.8 7.9 32 64PD years 2.3 1.2 0.5 6.0Hoehn&Yahr 1.5 0.5 1.0 2.5UPDRS motor score 17.1 5.1 6 25UPDRS tremor ipsilateral arm 0.9 0.9 0 2UPDRS rigidity ipsilateral arm 1.9 0.7 0 3Tremor power ipsilateral arm 2.4 1.4 0 7.4[123I]β-CIT binding contralateral striatum 2.3 0.7 1.3 4.2

*17 males and 13 females.Ipsilateral = the body side most affected by tremorPD years = duration Parkinson’s disease in yearsUPDRS motor = motor section of UPDRS

Figure 1. Power spectrum from a PD patient with tremor of dominant peak frequency at 6 Hz. The power of the movement frequency of the arms is presented in arbitrary units (au).

Chapter 2.1

24

Tremor and rigidity

Tremor power values are used as measure for tremor. The rigidity asymmetry index (RA) corresponded significantly with tremor power asymmetry index (p=0.002*). Figure 2 shows that the arm with a higher intensity of tremor is also more affected by rigidity.

A significant negative correlation was found between tremor power and UPDRS rigidity score in the ipsilateral arm (R=-0.48; p=0.01*; figure 3).

Figure 2. UPDRS rigidity asymmetry index (RA) versus tremor power asymmetry index (PA).

Figure 3. UPDRS rigidity scores versus relative tremor power of the ipsilateral arm. The UPDRS scores are expressed in points (0–4).

UPDRS rigidity asymmetry index

3,02,01,00,0-1,0-2,0-3,0

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Rigidity and [123I]β-CIT SPECT

Rigidity (RA) and SPECT (SA) asymmetry indices showed a significant relationship (p=0.001*; figure 4), indicating a relationship between the more affected body side on the basis of rigidity and the more severely impaired contralateral striatal [123I]ß-CIT binding. The correlation between UPDRS rigidity score of the ipsilateral arm and the contralat-eral striatal [123I]ß-CIT binding approached the level of significance (R=-0.32; p=0.08). ANOVA with rigidity as factor (three severity categories) showed that rigidity accounted for a significant part of the variance in the contralateral striatum (R2=0.28; p=0.01*).

Tremor and [123I]β-CIT SPECT

The SPECT (SA) asymmetry index showed a significant correspondence with the tremor power asymmetry index (PA; p=0.004*), indicating that the arm with a higher intensity of tremor coincided with the more severely impaired contralateral striatum. Figure 5 shows that in the majority of patients tremor appears contralateral to the brain side with the lowest striatal [123I]ß-CIT binding. Tremor power in the ipsilateral arm and the contralateral striatal [123I]ß-CIT binding did not show a significant relationship (R=0.19; p=0.31; figure 6). ANOVA analysis with tremor power as a factor (five severity categories) showed that tremor accounted for a significant part of the variance in the contralateral striatum (R2=0.37; p=0.02*). ANOVA with ipsilateral rigidity as factor (three severity categories) and ipsilateral tremor power as covariate (R2=0.29) showed that only rigidity was significantly related to the contralateral striatum (p=0.02*2), while tremor was not (p=0.5). No significant correlation was found between rigidity-cor-rected tremor power score (Z-score) in the ipsilateral arm and the contralateral striatal

Figure 4. Striatal ligand binding asymmetry index (SA) versus UPDRS rigidity asymmetry index (RA).

Figure 5. Striatal ligand binding asymmetry index (SA) versus tremor power asymmetry index (PA).

Striatal ligand binding asymmetry index

,6,4,2-,0-,2-,4-,6

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Chapter 2.1

26

[123I]ß-CIT binding (R=-0.2; p=0.3). ANOVA with ipsilateral rigidity-corrected tremor power score (Z-score) as factor (five severity categories) showed that the above men-tioned significant contribution of tremor in the variance in the contralateral striatum disappeared when corrected for the influence of rigidity (p=0.31).

Discussion

The majority of recent PET and SPECT imaging studies in PD patients show signifi-cant correlation between both the striatal [18F]fluorodopa and [123I]β-CIT binding, and the UPDRS scores for bradykinesia37,57 and rigidity58,59, but not for tremor. A previous [18F]fluorodopa PET study66 demonstrated the association between tremor as an iso-lated PD manifestation and reduced [18F]fluorodopa binding. Here, we investigated the relationship between tremor and the disruption of the dopaminergic pathway in PD patients, taking into consideration possible influence of factors such as medication effects and rigidity. A sensitive method of resting tremor assessment, accelerometry, was used. We found that: (1) in de novo PD patients, the dominant tremor and rigidity appear in the same arm, contralaterally to the more affected striatum; (2) tremor power accounted for a significant part of variance in the contralateral striatum, suggesting a relation between this PD symptom and the degeneration of the dopaminergic system; (3) rigidity significantly influenced the expression of tremor in the same arm; and (4) by correcting for the influence of rigidity, the significant contribution of tremor in the variance in the contralateral striatal [123I]β-CIT binding disappeared.

While our data do not demonstrate a direct relationship between tremor inten-sity and the dopaminergic dysfunction, they do not rule out the contribution of nigros-triatal dopaminergic system in the development of resting tremor. Our results show

Figure 6. Ligand binding ratio in the contralateral striatum versus relative tremor power (expressed as relative power of the tremor peak established by dividing the power of the tremor peak by the mean power of the total spectrum) of the ipsilateral arm.

Ligand binding contralateral striatum

(R=0.19; p=0.31)

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a correlation between the body side with the highest degree of tremor and the brain side with the severest dopaminergic dysfunction. This, together with tremor account-ing for small but significant part of variance in the contralateral striatum, suggests a possible relationship between those two variables. However, we also found that ri-gidity reduces the intensity of resting tremor. A detailed investigation of tremor power revealed a large variation of tremor power in PD patients with relatively minor rigidity, whereas a small variation in tremor power was observed in PD patients with a more substantial rigidity. The degree of tremor was reduced with increasing rigidity. In the group of patients with relatively high rigidity scores for the separate limbs, tremor power was considerably reduced compared with the highest tremor powers observed at the low rigidity scores. The fact that rigidity and tremor can occur within the same patient in the same extremity, contralateral to the more affected striatum, and that tremor is apparently suppressed in patients with high rigidity scores may contribute to our understanding of the nature of the relationship between the degree of degeneration of the dopaminergic pathway and the degree of tremor. Theoretically, the reported rela-tionship between resting tremor and rigidity allows for a further understanding of the nature of resting tremor in PD. Akamatsu and colleagues67 suggested that the tremor oscillations can be modeled from the perspective of nonlinear dynamics. Applying the logistic equation, a systematic variation of one control parameter (e.g. the degree of muscle activation) can result in changes between different mammalian types of tremor; that is, a transition from a point attractor (one stable point), to a limit cycle oscilla-tor (oscillator between two or more points), and even chaotic behaviour35,46. Resting tremor may be modeled by a nonlinear oscillator with stiffness (degree of rigidity) as an important control parameter68. Future studies should relate possible model param-eters to central and peripheral control mechanisms.

The significant influence of the rigidity on tremor expression supports the clini-cal distinction between the tremor type and the akinetic-rigid type PD patients. It is even temping to speculate that L-DOPA treatment in PD, which alleviates rigidity, might subsequently facilitate the appearance of resting tremor in some patients. Fur-ther research on this is necessary.

Clearly, it is essential for studies investigating tremor in PD patients to correct for the influence of rigidity. However, the present study design shows some limitations in establishing full disclosure of a relationship between tremor and dopaminergic de-generation. One of them is the assessment of the resting tremor only as a sitting task. Tremor registration during the performance of other tasks (e.g. standing or walking) might allow for a better understanding of the nature of resting tremor in PD. Sec-ondly, the patient population investigated in the present study comprises non-medi-cated, de novo PD patients with a relatively short duration of the disease. Findings are limited by the relatively small range of symptom severity (highest UPDRS tremor score was 2). However, we have accepted this disadvantage, because the homogeneity of this patient group concerning disease parameters was high and the effects of the

Chapter 2.1

2�

medication did not interfere with the interpretation of the observations. Also, it is neither ethical nor realistic to withhold patients in a later stage of the disease from medication. Furthermore, in order to cope with a problem of a limited range of UP-DRS tremor scores, the accelerometer recordings of resting tremor were performed, showing a broad range of tremor powers.

In conclusion, both tremor and rigidity appear in the same limb in early, non-medicated PD patients. Rigidity influences the expression of resting tremor, which refines the classical distinction between the tremor type and akinetic-rigid type of PD. The appearance of dominant tremor and rigidity in the same arm, contralateral to the more affected striatum, makes it important to correct for the influence of rigidity in studies were the relation between severity of tremor and dopaminergic dysfunction is investigated. Likewise, medication is most likely a confounding factor in finding such a relationship. However, the results of the present study show that when the rigidity is being taken into account the association between tremor and radioligand binding can not be demonstrated in non-medicated PD patients, suggesting that other pathophysi-ological mechanisms might be involved in the development of tremor.

CHAPTER 2.2

Rigidity and bradykinesia reduce interlimb coordination

in Parkinsonian gait

Adapted from A Winogrodzka, RC Wagenaar, J Booij, ECh Wolters

Archives of Physical Medicine and Rehabilitation 2005;86:183–189

Chapter 2.2

30

Summary

The exact pathogenesis of gait disturbances in Parkinson’s disease (PD) is still unclear. Only a few studies investigated this subject in patients after the withdrawal of medica-tion. In the present study we hypothesized that neurological symptoms such as brady-kinesia and rigidity limit the ability to change the coordination of arm and leg move-ments during gait when systematically manipulating walking speed in non-medicated PD patients. Furthermore, we hypothesized that this reduced flexibility of coordination is related to the extent of nigrostriatal dopaminergic degeneration. [123I]β-CIT SPECT was used to determine the extent of degeneration of the dopaminergic system. The dynamical systems approach was used to examine the interlimb coordination during systematic manipulation of walking speed on a treadmill in 29 early, de novo PD pa-tients. The phase relations between arm and leg movements were related to the clinical measures of rigidity and bradykinesia.

Significant correlations were found between the rigidity and bradykinesia and the coordination measures as well as contralateral striatal [123I]β-CIT SPECT binding and coordination measures, in terms of asymmetry indices.

Our findings indicate that early-stage PD patients are generally able to adapt their coordination patterns when walking speed was systematically manipulated. However, bradykinesia and rigidity as well as the extent of degeneration of the dopaminergic sys-tem were associated with a limited adaptive ability (flexibility) in movement coordi-nation. The combination of a drug treatment that controls bradykinesia and rigidity and a physical therapy exercise programs possibly using external cues mechanisms are required in order to obtain relevant effects on gait in PD patients.

31

Rigidity and bradykinesia reduce interlimb coordination in Parkinsonian gait

Introduction

Parkinson’s disease (PD) is a severe and progressive neurodegenerative disorder with bradykinesia and rigidity as well as tremor at rest69 and postural instability. Those symp-toms are associated with gait disorders characterized by shuffling steps, low walking speed, small stride length, reduced arm swing, rigidity in trunk movements, propulsion and retropulsion70,71. Despite use of dopaminergic medication patients can still face a re-lentless deterioration in mobility, which can eventually result in need for custodial care. It is generally accepted that the main features of PD result from a loss of dopaminergic cells in the substantia nigra and subsequent deficiency of dopamine in the striatum72. The exact pathogenesis of gait abnormalities in PD, however, is complex and still un-resolved. Different combinations of impaired neuronal control associated with rigidity, bradykinesia and postural control are thought to be involved70,73. Our study focuses on the relationship among neurological symptoms, the degeneration of the dopaminergic system, and the coordination of walking in PD.

The majority of research on locomotion in PD has focused so far on kinematic, ki-netic and electromyographic changes in the extremities. An overall lower speed, smaller stride length and reduced flexion and/or extension ranges in the joints of upper and lower extremities are typical features of gait in PD patients70,71,74,75. Morris et al76-79 sug-gested that PD patients with gait hypokinesia generally are unable to regulate stride length, while the ability to modulate cadence is retained. In their opinion gait hypoki-nesia is directly related to the inability to produce sufficiently large steps. Morris76-78 also suggested that attentional strategies and visual cues may facilitate the normal stepping pattern. Research on the coordination of bimanual movement and gait indicated that PD patients are limited in their ability to “switch” between coordinative patterns in or-der to meet changing environmental demands80,81.

In our study we used the dynamic systems approach to examine the changes in co-ordination patterns in PD patients82,83. The previous data suggest that in the human walk-ing, different coordination patterns exist, based on the relative coordination between arm and leg movements and between pelvic and thoracic rotations35. Those different coordination patterns, expressed as the relative phase between body segments (order pa-rameters), may be induced by manipulation of a single control parameter, such as walk-ing speed. Walking speed has been found an important control parameter in walking and can be used as basis for the evaluation of normal and pathological gait35. Gradually increasing walking speed results in important transitions in arm and leg movements in the normal human walking mode, which are often observed in the speed range 0.8–1.0 m/s35. Those changes in movement coordination can be evaluated by means of relative phase between body segments.

Van Emmerik et al83,84 reported overall less adaptations in coordination patterns between arm and leg movements as well as between pelvic and thoracic rotations when systematically varying walking speed in PD patients than healthy subjects. This reduced

Chapter 2.2

32

flexibility of adaptation during walking in PD patients appeared to be accompanied by an increased stability within coordination patterns. It is not clear whether this is directly related to neurological symptoms such as bradykinesia and rigidity. Bradykinesia might affect the adaptation of coordination pattern to changing walking speed. Rigidity has been found to affect single upper limb movements in PD patients85,86; however, it is un-clear how rigidity, as assessed at a single joint, transfers to situations involving whole body movements and patterns of interlimb coordination.

The extent and the pattern of the degeneration of the dopaminergic system in PD are described more extensively. Ouchi et al87 studied positron-emission tomography (PET) dopamine transporter radioligand uptake in the dopaminergic projection after gait and suggested that the reduced dopamine transporter availability in PD might be responsible for some aspects of the pathophysiology of gait in PD patients. Single-pho-ton emission computed tomography (SPECT) using the 123I-labelled cocaine analogue ß-CIT not only shows a significant loss of striatal dopamine transporters in PD patients as compared with controls52,54 but also significant correlations between the reduction in striatal ligand uptake in PD patients and the severity of symptoms, such as brady-kinesia and rigidity, as well as asymmetry in tremor expression between left and right arms37,42,55,69. At this moment, [123I]β-CIT SPECT is considered a highly reliable and re-producible imaging technique in PD40,88.

In our study, it is hypothesized that bradykinesia and rigidity are associated with the reduced ability to change the coordination of arm and leg movements (reduced flexibility) when systematically manipulating walking speed in PD patients and that this reduced flexibility during walking is related to the extent of dopaminergic degener-ation. To test these hypotheses, 29 early, non-medicated PD patients were investigated. Relative phase relations between arm and leg movements were calculated to study the flexibility of the coordination of walking. The severity of the neurological symptoms (rigidity and bradykinesia) was expressed by means of the Unified Parkinson’s Disease Rating Scale (UPDRS)89. [123I]β-CIT SPECT was used to determine the extent of degen-eration of the dopaminergic system.

Methods

Patient population

Twenty-nine early and drug naive PD patients (16 men, 13 women) with a mean age of 52.9 years were examined, 13 patients having unilateral and 16 patients having bilateral symptoms. All patients were diagnosed according to the criteria of the UK Parkinson’s Disease Society Brain Bank61, and showed a positive apomorphine chal-lenge test (subcutaneously injected dose of 3 mg), defined as 20% reduction or more in the UPDRS motor score, strengthening the diagnosis of PD62. The patients were recruited from the movement disorders unit of our outpatient clinic and gave writ-

33


ten informed consent to the research protocol. The Medical Ethical Committee of the Vrije Universiteit Medical Center gave permission for the study.

Study design

A single investigator (AW) performed the clinical assessment of all patients. The stage and severity of illness were assessed by means of the Hoehn and Yahr (H&Y) Staging Scale90 and UPDRS89.

The patients were instructed to walk on a treadmill (Techno Gym, RunRacer) with computer-controlled speed. During the experiment the speed was gradually in-creased from 0.6 km/h to the maximal attainable speed of 5.4 km/h, with 6 speed in-crements of 0.8 km/h, and then decreased again in similar steps. Each speed level was maintained for about one minute (total trial duration was 7 minutes), and the move-ments of the arms and legs were recorded for 30 seconds. Movements of the arms and legs in the sagittal plane were recorded with uniaxial accelerometers (Coulbourn type T-45) attached at the arms over the radial styloid and at the legs to the distal part of the medial tibia. The accelerometer signals were amplified through a transducer coupler (Coulbourn A-s72-25). Subsequently, SPECT experiments were performed. [123I]β-CIT was injected intravenously at an approximate dose of 110 MBq after treatment with potassium iodide to block thyroid uptake of free radioactive iodide. SPECT image ac-quisition was performed 24h post-injection63.

SPECT camera

The Strichman Medical Equipment 810X system was used for SPECT imaging. The transaxial resolution of this camera is 7.6 mm full width at half maximum of a line source in air. The energy window was set at 135-190 keV. 123I labeling was performed by Amersham Cygne (Technical University Eindhoven, The Nederlands), using the trimethylstannyl precursor of ß-CIT (specific activity >185MBq/nmol; radiochemical purity >99%) obtained from Research Biochemicals International (Natick, MA, USA).

AssessmentCoordination

Interlimb coordination during walking was evaluated by studying the (continuous) relative phase relations between movements of arms and legs for the right and left body side separately (right arm versus right leg and left arm versus left leg)64,83. The raw signals of the individual body segments were filtered with a low-pass Butter-worth 2nd order frequency with a cut-off frequency of 5 Hz, and, subsequently, the first derivative of the four filtered signals was obtained. To eliminate amplitude effects on relative phase measurement, both (filtered) acceleration and derivative of acceleration signals were normalized to the minima and maxima (-1 and 1) within

Chapter 2.2

34

each speed interval. The movements of each limb segment were described by a pair of phase variables, i.e.:

a_{s}(t)=r_{s}(t)cos[S_{s}(t)]

ast =r

st cos [S

st ] (1)

d_{s}(t)=r_{s}(t)sin[S_{s}(t)]

dst =r

st sin [S

st ] (2)

where as is the acceleration signal for body segment s, ds the derivative of acceleration, rs the amplitude, Ss the original signal, and t time. On the basis of these two phase vari-ables the phase angle was determined for each limb segment by means of the equation:

%fi_{s}(t)=arctan lbrace d_{s}(t)/a_{s}(t) rbrace

st =arctan {d

st /a

st } (3)

where φ is the phase angle. The phase angles were calculated in the range 0-180o degrees. All stride cycles were normalized to the shortest stride cycle which allowed superpositioning of stride cycles within one walking speed condition. The continuous relative phase between two segments was calculated by subtracting the phase angle of one segment from the phase angle of another segment for each point in a stride cycle91. The range of phase relations between arm and leg movements was estimated by calculating the mean relative phase (RP) at the highest achieved speed step for the right side (MAXRP-R) and the left side (MAXRP-L), separately. This parameter is dependent on the flexibility in movement coordination and on the number of accom-plished speed steps. The more speed steps the patient accomplishes, the higher the MAXRP. In addition, the change in relative phase with increasing speed was estimated for each patient by calculating the regression coefficients of the relative phase on speed for right and left body side, separately (COEFFRP-R and COEFFRP-L, respec-tively). The goodness of fit of the regression lines for each patient was adequate. The explained variance (R2) of the right-sided regression models was 0.64 (sd=0.28), of the left-sided models 0.67 (sd=0.30).

Not each patient could accomplish all seven speed steps. Therefore additional analyses were carried out after dividing the patients into 2 groups, that is: 1) patients accomplishing seven steps (group 1), and 2) patients accomplishing less than 7 steps (group 2). To assess the left-to-right asymmetry of the arm and leg coordination during walking, an asymmetry index (AI) was calculated for the above mentioned test parameters (MAXRP-AI and COEFFRP-AI), using the following formula:

AI= {"Left"-"Right"}over{"Left"}

AI=Left−Right

Left (4)

35


UPDRS

The rigidity score was calculated as the summed UPDRS rigidity in the arm and leg. The bradykinesia score was calculated as the summed score of the UPDRS items: facial expression, finger taps, hand movements, pronation-supination of the hands, leg agility, arising from chair and body bradykinesia89. Both scores were calculated for the right and left side, separately. To express the left-to-right asymmetry of rigidity (RIGAI ) and of bradykinesia (BKAI) an asymmetry index was calculated using Formula 4. To avoid dividing by zero all rigidity scores were increased by 1.

SPECT analysis

Slices were acquired during 300 s periods from the orbitomeatal line to the vertex, us-ing an interslice distance of 10 mm65. Data acquisition took place in a 128 x128 matrix. All images were corrected for attenuation as earlier described65. Reconstruction was carried out according to the manufacturer’s protocol package. The measured concentra-tion of radioactivity was expressed as Strichman Medical units (SMU’s; 1 SMU = 100 Bq/ml). For analysis of striatal [123I]ß-CIT binding, two transverse slices representing the most intense striatal binding were summed. A standard region of interest (ROI) template, constructed according to a stereotactic atlas and including regions for stria-tum and occipital cortex, was placed bilaterally on the combined image65. Estimates of specific striatal binding were made by subtracting occipital counts from striatal counts. The ratio of specific to non-specific striatal [123I]β-CIT binding was calculated, using the following formula63:

[I lsup 123 ]%beta"-"CIT binding={ROI - OCC} over {OCC}

[ I123

] -CIT binding=ROI−OCC

OCC (5)

in which ROI represents the mean radioactivity (in SMU) in the ROI (striatum), and OCC depicts the mean radioactivity in the occipital cortex. In order to express the left-to-right asymmetry of dopaminergic degeneration in PD patients the striatal [123I]β-CIT binding asymmetry index (SAI) was calculated using formula 4.

Statistical analysis

Variables not normally distributed were non-parametrically analyzed. Relationships be-tween variables were examined with Spearman’s rho. Means of independent subgroups were analyzed by means of the Mann-Whitney U test; in case of related samples the Wilcoxon Signed Rank Test was used. The 1-sample t-test was used to examine whether the change of the relative phase with increasing walking speed (COEFFRP) deviated sig-nificantly from zero. In case of multiple comparisons, a Bonferroni correction was used. An alpha of 0.05 was taken as the level of significance. All analyses were carried out with SPSS for Windows 9.0 software.

Chapter 2.2

36

RESULTS

The clinical and demographic data of 29 PD patients are presented in table 1. The coor-dination and SPECT data are presented in table 2. Correlations between clinical vari-ables, SPECT data and coordination measures are reported in table 3.

Table 1. Clinical and demographic data of 29 PD patients.

Mean SD Min Max

age 52.9 8.1 32 64

disease duration 2.5 1.4 0.5 6

H-Y stage 1.7 0.6 1 2.5

UPDRS-III 17.03 5.12 6 25

BK-R 7.6 3.5 1 13

BK-L 5.3 3.5 1 14

RIG-R 2.2 1.6 0 5

RIG-L 1.4 1.4 0 5Abbreviations: H&Y stage, Hoehn and Yahr stage; UPDRS-III, UPDRS total motor score ; BK-R, UPDRS bradykinesia score right side; BK-L, UPDRS bradykinesia score left side; RIG-R, UPDRS rigidity score right side; RIG-L, UPDRS rigidity score left side.

Table 2. Coordination and SPECT data of 29 PD patients

Mean SD Min Max

MAXRP-L 118.07 19.99 83.54 149.31

MAXRP-R 109.62 21.53 73.73 152.45

COEFFRP-L 6.99 5.46 -3.48 22.29

COEFFRP-R 4.09 4.87 -5.48 11.17ß-CIT binding left striatum 2.53 0.91 1.35 5.40

ß-CIT binding right striatum 2.68 0.87 1.33 4.80

Abbreviations: MAXRP-L, relative phase between left arm and left leg at the fastest speed step; MAXRP-R, relative phase between right arm and right leg at the fastest speed step; COEFFRP-L , regression coefficient of the regression of the relative phase between left arm and left leg on speed; COEFFRP-R, regression coefficient of the regression of the relative phase between right arm and right leg on speed.

Table 3. Correlations between clinical variables, SPECT data, and coordination measures.

Right side Left side

VariablesMAXRP COEFFRP MAXRP COEFFRP

rs p rs p rs p rs p

BK -0.61 <0.001 -0.34 <0.033 -0.59 <0.001 -0.38 <0.04

RIG -0.65 <0.001 -0.44 <0.016 -0.43 <0.022 -0.32 <0.096ß-CIT binding con-tralateral striatum 0.26 <0.18 0.26 <0.18 0.52 <0.09 0.12 <0.55

Abbreviations: MAXRP = relative phase between arm and leg at the highest achieved speed step; COEFFRP = regression coefficient of the regres-sion of the relative phase between arm and leg on speed; BK = UPDRS bradykinesia score; RIG = UPDRS rigidity score.

37


The maximal Hoehn-Yahr score was 2.5; the mean severity of the motor signs as as-sessed with the UPDRS section III was 17.03±5.12 (minimum-maximum, 6–25), and the mean disease duration was 2.5±1.4 years (minimum-maximum, 0.5–6) (table 1). In 20 patients, the most affected body side was the right side; in 9 patients, it was the left side. In all cases, the most affected side was the side with the first presentation of the disease.

Effects of walking speed

Nineteen patients accomplished all 7 increasing speed steps (speed group 1). Ten pa-tients accomplished fewer than 7 speed levels (speed group 2: 2 patients stopped after the 2nd level, 1 patient after the 4th level, 2 patients after the 5th level, and 5 patients after the 6th level).

Figure 1 depicts the changes in the mean relative phase between arm and leg movements on the right and left body sides as a function of increasing walking speed in the whole group of PD patients. The COEFFRP-L and COEFFRP-R were significantly larger than zero (p=0.0001). The increase of the relative phase with increasing speed (COEFFRP) as well as MAXRP were on the most affected body side significantly lower than on the opposite side (p<0.002 and p<0.001, respectively).

Marked individual differences in adaptability of relative phase when manipulat-ing walking speed were observed in PD patients, varying from very little adaptation to a gradual increase of relative phase with increasing speed. No significant correlations were found between age or sex, and coordination variables (MAXRP, COEFFRP) for ei-ther side of the body. No significant correlations were found between the absolute asym-metry index of MAXRP and COEFFRP and the maximal accomplished walking speed (r=0.07; p=0.88 and r=-0.21, p=0.65, respectively).

Figure 1. Changes in the mean relative phase between arm and leg as a function of increasing walking speed. Legend: ∇: right side; Ο: left side.

speed steps

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110

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Chapter 2.2

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Effects of rigidity and bradykinesia

Both bradykinesia and rigidity scores were significantly higher for the most affected side in comparison with the opposite side (p<0.001). Significant correlations were found between the bradykinesia and rigidity asymmetry indices and the asymmetry indices of MAXRP (r=-0.69, p<0.001 and r=-0.78, p<0.001, respectively) and COEFFRP (r=-0.47, p=0.01 and =-0.49, p=0.007, respectively; figure 2). Furthermore, significant correla-tions were found among rigidity, bradykinesia and both coordination measures, MAXRP and COEFFRP for the right side (r=-0.65, p<0.001 and r=-0.44, p=0.016, respectively; r=-0.61, p<0.001 and r=-0.34, p=0.033, respectively; see table 3). For the left body side, significant correlations were found among rigidity, bradykinesia, and, MAXRP (r=-0.43, p=0.022 and r=-0.59, p=0.001, respectively). The correlations among rigidity, bradyki-nesia, and COEFFRP approached the level of significance (r=-0.32, p=0.096 and r=-0.38, p=0.04; see table 3).

,6,4,2-,0-,2-,4-,6

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Figure 2. UPDRS rigidity and bradykinesia versus coordination measures (A) MAXRP and (B) COEFFRP expressed as asymmetry indexes (AI) (AI=(left-right)/left).

Legend: +: bradykinesia; Ο: rigidity.

3�


Striatal β-CIT binding versus rigidity and bradykinesia

Contralateral striatal ß-CIT binding (side opposite that of first presentation of motor signs) was significantly lower in comparison with the ipsilateral striatal ß-CIT binding (p<0.001). Significant correlations were found between the striatal ß-CIT binding and contralateral rigidity and bradykinesia (r=-0.54, p=0.001 and r=-0.54, p=0.003 for rigidity, and r=-0.58, p=0.001 and r=-0.49, p=0.008 for bradykinesia, for the right and left body side respectively).

Striatal β-CIT binding versus flexibility of walking patterns

There was a significant correlation between the asymmetry indices of MAXRP and COEFFRP and the striatal ß-CIT binding asymmetry indices (r=-0.77, p<0.001 and r=-0.58, p=0.001, respectively; figure 3). No significant correlations were found between MAXRP and COEFFRP for the right and left body side and contralateral striatal ß-CIT binding (r=0.26, p=0.18 and r=0.26, p=0.18 for right side; r=0.52, p=0.09, and r=0.12, p=0.55 for left side; see table 3).

,4,2-,0-,2-,4-,6

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Figure 3. Striatal [123I]β-CIT SPECT binding versus coordination measures (A) MAXRP and (B) COEFFRP expressed as asymmetry indexes (AI) (AI=(left-right)/left)

Chapter 2.2

40

Difference between accomplishing 7 speed steps or less

No significant differences in age, gender, disease duration, rigidity and bradykinesia scores, asymmetry indices of MAXRP and COEFFRP, and contralateral striatal ß-CIT binding were found between group 1 and group 2 (table 4). UPDRS III score was in group 2 slightly higher than in group 1, but not statistically significant (group 1, 16.5±5.02; group 2, 18±5.4; p=0.3). When group 1 and group 2 were examined sepa-rately, the same pattern of differences was seen between the most affected side and the opposite side concerning rigidity, bradykinesia and striatal ß-CIT binding as well as co-ordination variables, as in the whole group of PD patients. MAXRP for the most affected body side in group 1 was significantly higher than in group 2 (p=0.045). The difference in COEFFRP for the most affected body side between group 1 and 2 was not statistically significant (p=0.67). In group 2, a significant speed effect for relative phase was seen for the least affected side (p=0.003), but not at the most affected side (p=0.23).

Discussion

This is one of the few studies investigating coordination of gait in drug naive, de novo PD patients. Our previous research indicated that systematic manipulation of walking speed identifies a reduced flexibility in the coordination of walking in PD patients. In this way less adaptation in the coordination between body segments and hyperstability of coordination was observed in PD patients as compared with healthy controls84. In our study, we hypothesized that both classical PD symptoms, such as rigidity and bra-dykinesia and the loss of presynaptic dopaminergic integrity, would be associated with a reduced flexibility of coordination patterns between arm and leg movements during walking even in early stage of the disease. We further hypothesized that the degree of

Table 4. Comparison between group 1 and group 2.

Sex Age PD-yrs UPDRS-III BR RIG MAXRP COEFFRP MAXAI COEFFAI STR

M F

Group 1 13 6 52.7(7.4)

2.3(1.0)

16.5(5.02)

8.9(2.6)

2.9(1.3)

107.03(14.04)

3.62(3.5)

0.19(0.1)

1.96(3.3)

2.36(0.8)

Group 2 3 7 53.3(9.8)

2.4(1.6)

18.0(5.4)

10(2.3)

3.1(1.2)

95.53(24.5)

2.51(6.3)

0.22(0.2)

1.53(2.1)

2.25(0.5)

P NS NS NS NS NS <0.045 NS NS NS NS

Note: values are mean (SD); (the clinical and coordination data are shown for the most affected side).Abbreviations: M, male; F, female; UPDRS-III, UPDRS total motor score; BR, UPDRS bradykinesia score; RIG, UPDRS rigidity score; MAXRP,

relative phase between arm and leg at the highest achieved speed step; COEFFRP, regression coefficient of the regression of the relative phase between arm and leg on speed; MAXAI = asymmetry index of the relative phase between arm and leg at the fastest speed step achieved; COEFFAI = asym-metry index of the regression coefficient of the regression of the relative phase between arm and leg on speed; STR = ß-CIT binding in contralateral striatum, NS, not significant.

41


asymmetry in movement coordination between body sides would influence the maxi-mal walking speed of individual PD patients.

We found that the mean relative phase between arm and leg movements increased significantly with walking speed in the whole group of PD patients. We found marked individual different patterns in the changes of relative phase while scaling the walking speed. Hausdorff et al92,93 reported severe impairment of the ability to regulate stride-to-stride fluctuations in walking in PD patients, especially in relation with freezing of gait. In our study, a minority of patients showed very little adaptation in relative phase with in-creasing speed. Some patients showed considerable asymmetry in adaptation which was related to the severity and asymmetry of the disease symptoms. However, no significant relation was observed between the asymmetry in coordination and the maximal walking speed of the individual patients. The latter finding may be caused by the experimental design as the maximal speed condition was set at 5.4 km/h.

Another important finding of the present study was that both rigidity and bradyki-nesia were significantly associated with the ability to adapt the coordination of arm and leg movements during walking in PD patients. The side with the more pronounced coor-dination deficits in terms of both relative phase at the maximal achieved walking speed and the regression coefficient of the relative phase corresponded with the side that was more affected by bradykinesia and rigidity. Also the degree of flexibility in gait coordina-tion correlated significantly with bradykinesia and rigidity. Based on these findings, we are investigating now the influence of dopamineregic medication on the changes in the coordination between body segments in PD patients. We hypothesize that adequate drug treatment will facilitate coordination between body segments coinciding with improve-ments in rigidity and bradykinesia.

In the present study, we also investigated the relationship between interlimb coor-dination abnormalities in PD patients and loss of dopaminergic integrity. No significant correlation was found between the coordination variables and the extent of dopaminergic degeneration, as measured by striatal ß-CIT binding. The possible compensatory changes in the nigropallidal dopamine pathway contributing to preservation of motor function in early stage of PD, as recently suggested by Whone et al94, might be responsible for that. The analysis of the asymmetry indexes did reveal correspondence between the body side with the more pronounced disorders in coordination and the brain side with the more severe dopaminergic dysfunction, suggesting a possible relationship between these two variables. For full disclosure of such relationship, however, a more precise method identifying striatal subregional involvement might be necessary. In agreement with the results of previous PET and SPECT imaging studies in PD patients we also showed significant correlations between the striatal radioligand uptakes and the UPDRS scores for bradykinesia and rigidity37,42,55.

The findings of our study indicate that early-stage PD patients are generally able to adapt their arm and leg coordination pattern during walking with externally manipulated speed. This is in agreement with the findings of Zijlstra et al95, suggesting that the basic co-ordination patterns are preserved in PD patients. However, rigidity and bradykinesia limit

Chapter 2.2

42

this adaptation significantly. Therapeutic benefit of gait training combined with external cueing techniques may be especially important in the early stage PD patients in which the ability to adapt the coordination pattern is affected relatively little.35,78.

Study Limitations

A possible limitation of our study was the assessment of the interlimb coordination during treadmill walking, although this method allows for a systematic manipulation of walking speed as a control parameter. Generalizing our treadmill walking findings to overground walking may be limited84. Also, the findings within our patient population are limited by the relatively small range of the severity and duration of the symptoms. It should be noted, however, that the homogeneity of this patient group with regard to disease parameters was high, and the effects of the medication did not interfere with the interpretation of the observations. Controlling for the effects of medication is important to understand the ef-fects of PD on gait. In most of the studies on gait coordination in PD, patients were either on dopaminergic medication or withdrawn for several hours; in the latter case, the with-drawal period might be too short for the effects of the medication to subside completely.

Not all PD patients in our study population completed all 7 speed steps in the walking trial (maximal speed, 5,4km/h). However, more detailed analyses showed that the only significant difference for the patients who did not perform all 7 steps was the lower relative phase at the maximal obtained speed step, which is consistent with the result of the relative phase being a function of increasing walking speed. The disease severity expressed by means of UPDRS motor score and striatal ß-CIT binding was slightly more pronounced in patients who could only perform at lower velocities, but this finding was not statistically significant. This, together with the previously described habituation differences, might have caused the patients to stop the trial earlier.

Conclusions

In conclusion, the results of our study show that early, nonmedicated PD patients are able to adapt their coordination patterns between arm and leg movements when walking speed is systematically manipulated. However, in PD, this adaptation is limited because of rigidity and bradykinesia, and is related to the degeneration of the dopaminergic sys-tem. Combinations of drug treatment to control bradykinesia and rigidity, and physical therapy exercise programs possibly using external cueing mechanisms96 are required to obtain relevant effects on gait in PD patients.

CHAPTER 3

Leads for the development of neuroprotective treatment

in Parkinson´s disease and brain imaging methods

for estimating treatment efficacy

JC Stoof, A Winogrodzka, J Booij, FL van Muiswinkel, ECh Wolters, P Voorn, HJ Groenewegen, J Booij, B Drukarch

European Journal of Pharmacology 1999;375:75–86

Chapter 3

44

Summary

Patients suffering from Parkinson’s disease display severe and progressive deficits in motor behaviour, predominantly as a consequence of degeneration of dopaminergic neurons, located in the mesencephalon, and projecting to striatal regions. The cause of Parkinson’s disease is still an enigma. Consequently, the pharmacotherapy of Parkin-son’s disease consists of symptomatic treatment, with in particular L-dihydroxyphenyl-alanine (L-DOPA) and/or dopamine receptor agonists. These induce a dramatic initial improvement. However, serious problems gradually develop during long-term treat-ment. Therefore, a more rational, c.q. causal treatment is needed which requires the introduction of compounds ameliorating the disease process itself. The development of such compounds necessitates (1) more information on the etiopathogegesis, i.e., the cascade of events that ultimately leads to degeneration of the dopaminergic neurons, and (2) brain imaging methods, to estimate the extent of the degeneration of the dopa-minergic neurons in the living patient. This is not only important for the early diagnosis, but will also allow to monitor the effectiveness of alleged neuroprotective compounds on a longitudinal base. In this paper, etiopathogenic mechanisms are highlighted along the line of the oxidative stress hypothesis and within this framework, attention is mainly focused on the putative role of glutathione, dopamine auto-oxydation and phase II biotransformation enzymes. Especially, drugs able to increase the activity of phase II biotransformation enzymes seem to elicit a broad-spectrum of (neuro)protective re-sponse, and provide very promising leads for the development of neuroprotective treat-ment strategies in Parkinson’s disease. New developments in brain imaging methods (single photon emission computed tomography (SPECT) and positron emission tomog-raphy (PET)) to visualize the integrity of the striatal dopaminergic neurons in humans are highlighted as well. Especially, the introduction of radioligands that bind selectively to the dopamine transporter seems to be a significant step forward for the early diagno-sis of Parkinson’s disease. Performing these brain imaging studies with fixed time inter-vals does not only create the possibility to follow the degeneration rate of dopaminergic neurons in Parkinsons’s disease but also provides the opportunity to estimate therapeu-tic effects of putative neuroprotective agents in the individual patient.

45

Leads for the development of neuroprotective treatment in Parkinson´s disease and brain imaging methods for estimating treatment efficacy

Introduction

1.1. Symptoms and pathofysiology of Parkinson’s disease

Idiopathic Parkinson’s disease is a progressive neurodegenerative disorder which mani-fests itself by bradykinesia in combination with rigidity, tremor and a postural imbalance. In addition, autonomic nervous system dysfunctions such as micturition problems, ortho-static hypotension and seborrhoea as well as subtle cognitive dysfunctions and depression occur. Parkinson’s disease affects approximately 0.15% of the total population but 0.5% of people older than 50 years. The disease is neuropathologically characterized primarily by degeneration of dopamine-containing neurons in the ventral mesencephalon.

Dopaminergic neurons in the ventral mesencephalon are distributed over differ-ent cell groups, including the substantia nigra pars compacta (A9 cell group), the ventral tegmental area (A10 cell group) and the retrorubral area (A8 cell group). Recent neuro-anatomical and functional studies have revealed that the dopaminergic projection is just one of the neuronal elements integrated in the basal ganglia-thalamocortical circuits which are involved in the regulation of motor and complex behavioural activity97-100.

The following brief description of the functional neuroanatomy of these basal gan-glia-thalamocortical circuits is useful in understanding the symptomatology of Parkin-son’s disease and the pharmacotherapy that is currently used.

The basal ganglia consist of four main structures: the striatum (caudate nucleus, pu-tamen and nucleus accumbens), the pallidum (external and internal segments of the glo-bus pallidus and ventral pallidum), the subthalamic nucleus and the substantia nigra (pars compacta and pars reticulata). The striatum is the input structure of the basal ganglia, receiving afferents, in a strict topographical way, from the entire cerebral cortex (including “cortical-like” nuclei of the amygdala and the hippocampal formation), the midline and intralaminar thalamic nuclei, and midbrain serotonergic and dopaminergic cell groups. The output structure of the basal ganglia consists of the internal segment of the globus pal-lidus and the pars reticulata of the substantia nigra which project, also in a topographical manner, to different medial and ventral thalamic nuclei, the deep layers of the superior col-liculus and the reticular formation of the mesencephalon. The various thalamic nuclei that are innervated by these output structures of the basal ganglia project to different cortical areas of the frontal lobe, including motor, premotor and prefrontal cortical areas.

The topography in the projections from different frontal cortical areas through the basal ganglia and the thalamus by subsequent corticostriatal, striatopallidal (or striatoni-gral), pallidothalamic (or nigrothalamic), and thalamocortical projections is organised in such a way that a number of parallel, functionally segregated basal ganglia-thalamo-cortical circuits can be discriminated. Whereas sensorimotor function are dealt with in the circuit that originates in and returns to the (pre)motor cortex (and that at the striatal level, receives also inputs from the somatosensory cortex), other circuits are involved in complex motor/behavioral, cognitive and affective processes97,101,102.

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The input (striatum) and output structures (internal segment of the globus pallidus and pars reticulata of the substantia nigra) of the basal ganglia are connected with each other by means of two pathways, i.e. a “ direct” and an “indirect” pathway. The direct pathway consists of the γ-aminobutyric acid (GABA)/substance P/dynorphin-containing striatopallidal (inter-nal segment of the globus pallidus) and striatonigral (pars reticulata of the substantia nigra) projections. The indirect pathway is constituted by the sequence of the GABA/enkephalin-containing striatopallidal (external segment of the globus pallidus), the GABA-ergic pallido-subthalamic, and the glutamatergic subthalamo-pallidal (internal segment of the globus pal-lidus) and subthalamo-nigral (pars compacta of the substantia nigra) projections. At the level of the output structures of the basal ganglia, these direct and indirect pathways have opposite effect on the GABA-ergic neurons that project to the thalamic nuclei, the superior colliculus and the reticular formation. A “balance” between these two striatal output pathways appears to be essential for the normal regulation of movement. Although recent data show a more complex picture of the distribution of dopamine receptors, it has previously been suggested that dopamine, acting through different dopamine receptors, has opposing effects on the di-rect and indirect pathways97,103,104: via dopamine D1 receptors a stimulatory effect on the direct pathway, whereas via dopamine D2 receptors an inhibitory effect on the indirect pathway. The consequence of the loss of dopaminergic input to the striatum as occurs in Parkinson’s disease is therefore thought to be an increase in the output from the internal segment of the globus pallidus and the pars reticulata of the substantia nigra to the thalamus105, ultimately (suppos-edly at about 75% depletion of striatal dopamine) resulting in a reduction of cortical activa-tion which accounts for (most of) the Parkinsonian signs, including bradykinesia, rigidity and postural imbalance.

1.2. Symptomatic treatment of Parkinson’s disease

‘Restoring the balance’ at the level of the output structures of the basal ganglia in order to de-crease the inhibition of the thalamic nuclei can be achieved neurosurgically by lesioning the subthalamic nucleus, a posteroventral pallidotomy and/or venterolateral thalamotomy102. However, this therapeutic intervention is only rarely applied. In general, Parkinson’s disease patients are treated pharmacologically by suppletion and/or substitution of dopamine with the dopamine precursor L-DOPA or with dopamine receptor agonists106. In the late sixties, shortly after it became apparent that patients with Parkinson’s disease were suffering from a dopamine deficit in the basal ganglia, the dopamine precursor L-DOPA (soon afterwards in combination with a peripheral decarboxylase inhibitor) was successfully given to sup-plete the empty dopamine stores. Currently, this treatment is still considered to be the most effective way to control the symptoms of Parkinson’s disease. Unfortunately, however, long-term treatment with L-DOPA frequently results in fading of the therapeutic effect (wearing-off), in the development of serious motor side-effects such as on–off motor oscillations and dyskinesias and, although less often, in psychiatric complications. Under those conditions, increasing the dose of L-DOPA to compensate for the loss of therapeutic efficacy gives rise

47


only to more side-effects without adding any beneficial effect. The mechanisms underlying this ‘narrowing of the therapeutic window’ are still largely a matter of speculation. Lately, the hypothesis has been put forward that long-term treatment with L-DOPA might accelerate the degeneration of dopaminergic neurons by enhanced generation of cytotoxic reactive ox-ygen species as a consequence of dopamine and/or L-DOPA auto-oxidation (see also below). From the 1980’s onwards, the introduction of dopamine D2 receptor agonists has extended the therapeutic armamentarium for Parkinson’s disease. Although long-term treatment with dopamine D2 receptor agonists results in less dyskinesias, the therapeutic efficacy is likewise less dramatic as compared to the initial effects of L-DOPA. Furthermore, increasing the dose of dopaminergic agonists gives rise to other serious side-effects, such as psychotic reactions. Based on the above-described insights into the clinical action of the drugs, a now generally accepted therapeutic protocol consists of the combination of a low dose of L-DOPA together with one of the dopamine D2 receptor agonists. This treatment regime in general results in optimal control of the symptoms with fewer side-effects, at least in the early stages of the disease. Nevertheless, in the medium to long-run also this therapeutic strategy is doomed to failure. Apart from dopamine D2 receptors, also dopamine D1 receptors are targets for the action of dopamine in the striatum. Especially, the (medium spiny) output neurons project-ing to the internal segment of the globus pallidus, forming the so-called ‘direct route’, are known to express significant numbers of dopamine D1 receptors, whereas the cells of the ‘indirect route’ are supposed to contain predominantly dopamine D2 receptors104,107. Dopa-mine, by simultaneously facilitating the activity of the ‘direct’ pathway (via stimulation of do-pamine D1 receptors) and inhibiting the activity of the ‘indirect’ pathway (via stimulation of dopamine D2 receptors), keeps these pathways in a delicate balance which, as noted above, is thought to be essential for the normal regulation of movement. Moreover, this concept pro-vides also a rationale for the observed requirement of both dopamine D1 and dopamine D2 receptor stimulation to restore the complete repertoire of motor activity in animal models for Parkinson’s disease108. The fact that dopamine D2 receptor agonists mediate therapeutic effects on their own, especially in the early phases of Parkinson’s disease, could mean that in that stage residual dopamine released from surviving dopaminergic neurons is still sufficient to stimulate the ‘direct’ pathway109. Thus, one would expect selective dopamine D1 receptor agonists to be of additional therapeutic value later on in the course of Parkinson’s disease. However, in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-lesioned monkey model of Parkinson’s disease, the partial dopamine D1 receptor agonist SKF 38393, which was developed in the late seventies, failed to stimulate motor behavior110,111. Recently, it was demonstrated that at least in primates, this compound’s lack of therapeutic effect is most likely due to its very low intrinsic activity at the dopamine D1 receptor112,113. Subsequently, full dopamine D1 receptor agonists became available and animal studies have now definitely proven that stimulation of dopamine D1 receptors affects motor behaviour, especially in those animals in which the nigrostriatal dopaminergic system has been lesioned114-116. How-ever, careful analysis of this behavioural stimulation casts doubt on the anti-Parkinsonian nature of the effects of dopamine D1 receptor agonists. For example, the claim that dopamine

Chapter 3

4�

D1 receptor stimulation activates motor behaviour without inducing dyskinesias cannot be generally confirmed117,118. In fact, long-term stimulation seems to induce significant dyskine-sias. Another drawback of long-term treatment with dopamine D1 receptor agonists appears to be the poorly sustained action of these drugs because of receptor desensitisation117, and last but not least, the occurrence of seizures119-122. Thus, contrary to expectation, dopamine D1 receptor stimulation, even with selective high efficacy agonists, in our opinion, will not substantially improve the pharmacotherapy of Parkinson’s disease.

1.3. Urgent need for a more causal treatment

One of the mechanisms likely to underlie the occurrence of wearing-off and the develop-ment of serious side-effects upon long-term treatment with dopaminergic compounds in Parkinson’s disease is the progression of the pathologic process. Thus, the ongoing de-generation of dopaminergic neurons might not only prohibit efficient decarboxylation of the administered L-DOPA but could also be responsible for changes in (postsynaptic) dopamine receptor sensitivity, resulting in the aforementioned problems. Therefore, as of late, much effort has been invested in the development of neuroprotective agents that will be able slow down the degenerative process. In order to design an optimal causal treat-ment regime for Parkinson’s disease, two lines of investigation are of crucial importance. First, in-depth knowledge about the whole cascade of events that leads to the degenera-tion of the dopaminergic cells, i.e., the etiopathogenesis of Parkinson’s disease, is required (Section 2), since such knowledge is indispensable for the rational development of neu-roprotective agents. Secondly, in order to assess the effectiveness of such compounds in longitudinal studies, a brain imaging technique that visualizes the striatal dopaminergic innervation and is able to estimate the rate of degeneration of the dopaminergic system in the course of Parkinson’s disease (Section 3) should be available.

2. Etiopathogenesis of Parkinson’s disease

2.1. Oxidative stress hypothesis

As noted already, at the cellular level Parkinson’s disease is characterized foremost by a loss of the neurotransmitter dopamine in the striatum due to degeneration of dopami-nergic neurons located in the pars compacta of the substantia nigra123. In addition, also other neuronal systems, in particular noradrenergic neurons originating in the locus coeruleus, are affected by the disease process albeit to a substantially smaller extent124. Despite large scale attempts at elucidation, presently the pathogenesis of Parkinson’s disease remains largely enigmatic. Nevertheless, already for a number of years oxida-tive stress has been implicated as a major causative factor in the neuronal degeneration occurring in the Parkinsonian substantia nigra. For example, post-mortem analysis has revealed increased lipid peroxidation, superoxide dismutase activity and free iron lev-

4�


els in the substantia nigra of Parkinsonian patients125. However, the earliest, reportedly even preceding the loss of dopamine, indication of oxidative stress in Parkinson’s dis-ease brains appears to be a reduction in the level of the anti-oxidant glutathione in the substantia nigra126. Considering these findings and the ease with which the catechol de-rivate dopamine oxidizes, it has been postulated that the preferential degeneration of ni-gral dopaminergic neurons in Parkinson’s disease is related or might even be attributed to reactive oxygen species produced during dopamine breakdown. This line of thought has come to be known as the ‘oxidative stress’ or ‘free radical hypothesis of Parkinson’s disease127. In fact, the “classical”, i.e., monoamine oxidase-catalyzed, route of oxidative dopamine breakdown leads to the formation of the strong oxidant hydrogen peroxide. Consequently, based on the ‘oxidative stress hypothesis of Parkinson’s disease, it was ex-pected that pharmacological blockade of monoamine oxidase activity would offer neu-roprotection. Eventually, this led to the introduction of the monoamine oxidase inhibi-tor deprenyl for clinical use as an alleged neuroprotectant in Parkinson’s disease128,129. Unfortunately though, for a number of possible reasons, the potential to inhibit pro-gression of the disease process in humans (solely) via blockade of monoamine oxidase until now has proven to be rather disappointing130,131. Therefore, a different approach to oxidative stress and neuroprotection in Parkinson’s disease is clearly warranted.

2.2. Glutathione and the brain

There is now ample evidence suggesting that loss of glutathione in the substantia nigra is an important, if not primary, event in the pathogenesis of Parkinson’s disease. Glutathi-one is a tripeptide containing a glutamate, a cysteine and a glycine moiety. Glutathione is present in millimolar concentrations in all cells of eukaryotic organisms, including humans. Synthesis and degradation of glutathione occur in a number of highly inter-active enzymatic reactions known collectively as the ‘γ-glutamyl cycle’132. Extensively studied enzymes of this cycle are γ-glutamylcysteine synthetase and γ-glutamyltrans-peptidase. These enzymes are responsible for catalyzing the formation of the gluta-thione precursor γ-glutamylcysteine from glutamate and cysteine and the breakdown of glutathione into glutamate and cysteinylglycine, respectively. Cells use glutathione mainly as an anti-oxidant for the scavenging of organic- and inorganic hydroperox-ides, such as lipid peroxides and hydrogen peroxide, and for inactivation of endogenous toxic metabolites and xenobiotics through conjugation. These reactions are catalyzed by the enzymes glutathione peroxidase and glutathione transferase, respectively132. All components of glutathione metabolism have been shown to be present in the brain. However, the cellular localization in the brain of glutathione and related enzymes is still a matter of debate. Thus, using a rather nonspecific histochemical technique, glutathi-one was originally reported to be present almost exclusively in glial (astrocytic) cells133. This finding was supported by data obtained from cultured brain cells demonstrating a substantially higher content of glutathione in astrocytes as compared to neurons134.

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50

In contrast, with the use of specific antibodies to glutathione on tissue sections and primary brain cell cultures, it has recently been established that apart from astrocytes, glutathione is present in substantial amounts also in neuronal fibers135-137. A similarly unclear picture has emerged from studies investigating the presence of the various glu-tathione related enzymes in the different cell types of the brain. For example, whereas γ-glutamyl transpeptidase immunoreactivity has been detected solely in endothelial and ependymal cells, biochemical activity of this enzyme seems to be present also in other types of brain cells138-140. The same sort of confounding observations have been made regarding glutathione peroxidase and glutathione transferase139-141. In trying to explain these inconsistencies, it is important to notice that comparison of the results be-tween the various studies is complicated by differences not only in the type(s) of animal species and experimental methods used but also in the brain areas investigated.

2.3. Glutathione and Parkinson’s disease

The loss of glutathione in Parkinson’s disease is reported to be restricted to the substan-tia nigra, the brain area most severely affected by the disease process. More important, a decreased nigral glutathione content appears to be rather specific for Parkinson’s disease since other disorders involving the substantia nigra, such as multisystem atrophy, are not accompanied by such a deficit142,143. Furthermore, the reduction in glutathione content is accounted for solely by the reduced form of the peptide as no changes in the level of the oxidized form have been demonstrated142,143. In contrast to glutathione content, only scant information is available on the status of glutathione related enzymes in Parkinson’s disease brains. The data available, however, show no consistent change in any enzyme activity144,145. A significant increase in the activity of γ-glutamyl transpeptidase in the sub-stantia nigra of Parkinson’s disease patients was reported several years ago145. Since γ-glu-tamyl transpeptidase, which is a cell membrane bound protein, presumably functions to increase intracellular glutathione by retrieval of extracellular glutathione (or its compo-nent amino acids), apparently in Parkinson’s disease-afflicted brain, an attempt is made to compensate for the loss of intracellular glutathione by upregulation of γ-glutamyl trans-peptidase activity. Together, this combination of an early decrease in cellular glutathione content, an increase in glutathione ‘uptake’ from the extracellular space and an absence of overt alterations in the enzymatic capacity to synthesize or use glutathione for protec-tion against oxidative damage and/or toxic compounds suggests that the degeneration of nigral dopaminergic neurons in Parkinson’s disease may be caused by a, thus far unidenti-fied, process in which glutathione is consumed excessively. In order to possibly identify the nature of this ‘glutathione consumption’ process in Parkinson’s disease, it is important to take into consideration that most assays used for measuring glutathione content detect only the oxidized and/or reduced form of the compound but provide no information on other major forms in which the peptide can occur, for instance, glutathione conjugates. In fact, the increasing number of studies devoted to this topic show a rise in the ratio of

51


glutathionyl (i.e., cysteinyl) adducts of dopamine to dopamine or dopamine metabolites in both the substantia nigra and cerebrospinal fluid of Parkinson’s disease patients146-148. Since these adducts are thought to be formed largely as a consequence of the interaction between glutathione and so-called dopamine–quinones which are the foremost product of the nonenzymatic, auto-oxidative breakdown of dopamine (see below), these data point towards a possible link between the early glutathione loss observed in Parkinson’s disease and the process of dopamine auto-oxidation occurring in the substantia nigra.

2.4. Dopamine auto-oxidation and neuromelanin in the substantia nigra

In addition to the well-known monoamine oxidase-catalyzed enzymatic oxidation, do-pamine is also able to undergo oxidative catabolism in a chain of reactions designated as dopamine auto-oxidation. During this not yet unequivocally characterized process, dopamine is oxidized first into a product named dopamine–quinone which cyclizes readily to form an indolic compound which is known under various names of which aminochrome appears to be the one used most extensively149. Subsequently, this cyclic dopamine–quinone is oxidatively polymerized leading to the formation of neuromela-nin, the pigment which in adult humans lends the substantia nigra its characteristic dark brown to black color149,150. The pathway for generation of the neuromelanin poly-mer in vivo is more complicated than described here because other factors such as metal ions and sulfhydryl agents affect the chemistry involved. Curiously, dopamine auto-oxi-dation apparently does not occur at the same rate in all dopaminergic neurons since the amount of intracellular neuromelanin differs markedly between dopamine-containing cells in the substantia nigra. In this context, it is important to note that neuropathologi-cal studies in Parkinson’s disease have revealed that the loss of dopaminergic neurons is not distributed equally over the substantia nigra but occurs primarily in those cells heavily laden with neuromelanin granules151. Recently, these data, suggesting a posi-tive correlation between the extent of melanization and neuronal death in Parkinson’s disease, gained support from the observation of a low number of melanized neurons in the substantia nigra of a population with a relatively low prevalence of Parkinson’s disease152. Hence, in light of the evidence described in this and the previous section, it appears quite feasible to assume that the process of dopamine auto-oxidation in the substantia nigra may be directly associated to the pathogenesis of Parkinson’s disease.

2.5. Dopamine auto-oxidation as a source of oxidative stress in Parkinson’s disease

Whereas the functional significance of neuromelanin and its exact relation to Par-kinson’s disease pathogenesis is still open to question150 there is now conclusive evi-dence to illustrate that the process of dopamine auto-oxidation involves the formation

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of potentially cytotoxic dopamine intermediates and constitutes a major source of oxidative stress in the human substantia nigra153. Thus, as described, the auto-oxida-tion of dopamine yields dopamine–quinones, i.e., highly reactive, electron-deficient, compounds that are able to exert cytotoxic effects in a variety of ways, for instance, by causing glutathione depletion as a result of covalent binding of the quinone to the reduced sulfhydryl residue contained in the glutathione molecule154. Moreover, as shown recently, the resultant glutathionyl-dopamine adduct, although intended as a detoxified form of the dopamine–quinone, is prone to further oxidation into a highly neurotoxic benzothiazine derivative155. In addition to deleterious effects due to direct interaction with cellular constituents, dopamine–quinones are believed to be responsible for cytotoxicity also via their propensity for redox-cycling with the consequent generation of reactive oxygen species154. Thus, at the expense of cellu-lar reducing equivalents in the form of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and/or reduced nicotinamide-adenine dinucleotide (NADH) and catalyzed by quinone-reducing enzymes, in particular NADPH-cytochrome P450 reductase, the one-electron reduction of the dopamine auto-oxidation product ami-nochrome results in the formation of dopamine–semiquinone, a highly reactive and unstable dopamine intermediate that binds readily to cellular nucleophiles and/or reoxidizes to aminochrome with the concomitant production of superoxide radicals and hydrogen peroxide156,157. In this way, aminochrome reduction elicits a cascade of reversible oxidation and reduction reactions, or so-called redox cycle, that is accom-panied by excessive release of reactive oxygen species and depletion of NADPH. Via the iron (Fe2+)-catalyzed Haber–Weiss reaction, on their turn, the superoxide radicals and hydrogen peroxide released during aminochrome redox-cycling are able to yield the extremely toxic hydroxyl radical thereby initiating a variety of detrimental events ultimately leading to dopaminergic cell death158. These include lipid and protein oxi-dation, damage to nucleic acids, aberrant redox-sensitive gene transcription, depriva-tion of cellular energy production and apoptosis, phenomena which have all been observed to occur in the Parkinsonian brain125. Moreover, with respect to oxidative stress and Parkinson’s disease, a (dopamine–quinone-induced) deficiency of NADPH is noteworthy in that it leads to a general reduction of cellular protective anti-oxidant capacity. Thus, while the enzyme glutathione reductase utilizes NADPH to maintain glutathione in its reduced state, reduced glutathione is required for the catalytic ac-tivity of glutathione peroxidase which, as noted before, prevents the accumulation of both hydrogen peroxide and lipid peroxides132. NADPH loss will therefore lead to an impaired reduction of oxidized glutathione by glutathione reductase with a consequent failure of scavenging toxic peroxides by glutathione peroxidase. Taken together, there appears to be ample reason to suspect that the processes underlying dopamine auto-oxidation contribute greatly to the oxidative stress and subsequent neuronal damage as observed in the substantia nigra in Parkinson’s disease. Moreover, given the biochemical composition of neuromelanin and the occurrence of both the

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dopamine–quinone derivate 5-S-cysteinyl-dopamine and NADPH-cytochrome P450 reductase in human brain tissue, it is now evident that such a pro-oxidative pathway is indeed operative in vivo150,153,159.

2.6. Detoxication of dopamine auto-oxidation products by phase II biotransformation enzymes

Alongside the aforementioned pro-oxidant pathways, at least two anti-oxidant path-ways have been implicated in the detoxication of dopamine-derived quinones. These pathways depend primarily on the presence of reduced glutathione and/or the catalytic activity of a group of enzymes that are collectively known as phase II biotransforma-tion enzymes. Whereas phase I biotransformation enzymes, like the cytochrome P450 family of proteins, in general, increase the reactivity of their substrates and generate reactive oxygen species, phase II biotransformation enzymes, of which glutathione transferase, uridine 5′-diphosphate glucuronosyltransferase and sulfotransferase are probably the best known examples, have been shown to protect against xenobiotics and endogenous toxic metabolites by catalyzing the transformation of reactive electrophiles into more hydrophilic (inactive) conjugates that subsequently are excreted from the cell160,161. Originally, cellular defence against the toxicity of aminochrome and other dopamine–quinones was thought to be provided by the phase II biotransformation enzyme DT-diaphorase (NAD(P)H:quinone acceptor oxidoreductase), a flavoenzyme which, in contrast to NAD(P)H-cytochrome P450 reductase, catalyzes a two-electron reduction of aminochrome yielding dopamine–hydroquinone without the intermedi-ate formation of free dopamine–semiquinone162. On the condition that tissue anti-oxi-dant capacity, e.g., in the form of reactive oxygen species-scavenging enzymes such as superoxide dismutase and catalase, is sufficient to prevent its auto-oxidation, dopa-mine–hydroquinone is a redox stabile entity that lacks electrophilic reactivity and, due to the presence of hydroxyl groups at its terminal quinone moieties, fulfills the chemi-cal requirements for further biotransformation and detoxication by, for instance, uri-dine 5′-diphosphate glucuronosyltransferase or sulfotransferase158. Recently, apart from DT-diaphorase, μ class glutathione transferases, in particular the μ2-2 subtype, have been identified to constitute an additional protective mechanism against the toxicity of dopamine-derived quinones by catalyzing the reductive conjugation of glutathione to aminochrome. In this reaction, 4-S-glutathionyl-5,6-dihydroxyindole is formed which, in contrast to the glutathionyl adduct of the uncyclized dopamine–quinone, is a stable entity that is resistant to redox cycling and also amenable to further detoxication either by other phase II biotransformation enzymes or via direct cellular excretion156,163. Taken together, it seems therefore that under physiological conditions, the concerted action of phase II biotransformation enzymes, most notably DT-diaphorase and glutathione transferase, provides a powerful cellular defence mechanism against the oxidative stress inherent to dopamine auto-oxidation, whereas insufficient activity of this enzyme sys-

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tem may lead to oxidative damage such as observed in Parkinson’s disease. In this re-spect, it is noteworthy that while (immuno)histochemical and biochemical analysis of rat brain revealed the presence of DT-diaphorase in mesencephalic dopaminergic neu-rons and glial cells164,165, molecular biological techniques have shown also the abundant expression of μ class glutathione transferases, including the μ2-2 form, in human brain structures such as the substantia nigra163.

2.7. Phase II biotransformation enzymes as target for neuroprotection in Parkinson’s disease

Apart from a putative involvement in Parkinson’s disease pathogenesis158, the pivotal role of phase II biotransformation enzymes in the inactivation of dopamine-derived cytotoxic quinones and their presence in relevant brain structures, makes these anti-oxidant pro-teins attractive targets for the rational development of innovative, neuroprotective agents to treat Parkinson’s disease. Nevertheless, until now the therapeutic potential of manipu-lation of phase II enzyme activity is not widely acknowledged in neuroscience. This may at least in part be attributed to the lack of widespread information concerning the mech-anisms regulating phase II enzyme expression in the nervous system. In this respect, it is of note, that besides their capability to detoxify a broad spectrum of electrophilic substances, phase II biotransformation enzymes have in common their inducibility by a large variety of structurally diverse chemicals of both natural and synthetic origin161,166. In fact, these compounds, including phenolic anti-oxidants (e.g., butylated hydroxyani-sole, tea polyphenols and phytoestrogens)161,166,167 aromatic isothiocyanates (e.g., sulfora-phane)168, nonsteroidal anti-inflammatory drugs (e.g., indomethacin, ibuprofen)169, and many others, have been shown to confer cellular protection via a coordinated upregula-tion of the expression of phase II enzymes, most likely mediated by activation of a so-called anti-oxidant response element present in the promotor region of the respective genes170. In this context, a particularly interesting and well-studied class of compounds is the dithiolethiones, a cyclic, sulfur-containing group of agents, originally described as constituents of cruciferous vegetables171. The dithiolethiones, of which oltipraz and anethole dithiolethione are available for clinical use in humans172-173, not only increase the activity of phase II biotransformation enzymes in various cellular preparations in vitro174-176, but are active also in vivo in animals and humans with only minor side-effects reported171-173. In addition to their effect on phase II biotransformation, dithiolethiones are especially attractive in that they boost general cellular anti-oxidant capacity by acting as regular oxidant scavengers172, by increasing the expression of metal-binding proteins such as ferritin178, and by stimulation of the enzymes responsible for the maintenance of reduced glutathione pools, in particular γ-glutamylcysteine synthetase, glutathione reductase and glucose-6-phosphate dehydrogenase171,177,179,180. In fact, such an additional mechanism of action has recently been shown to contribute significantly to the capacity of phase II enzyme inducers to protect against dopamine neurotoxicity181.

55


Thus, given the likely role of dopamine auto-oxidation in Parkinson’s disease pathogenesis and the observation that drugs identified by their ability to increase the activity of phase II biotransformation enzymes are able to elicit a broad-spectrum (neuro)protective response, it seems plausible to conclude that such compounds are very promising leads for the development of efficacious neuroprotective treatment strat-egies in Parkinson’s disease.

3. Brain imaging techniques to determine the integrity of the dopaminergic system and to monitor its degeneration rate

3.1. SPECT studies with [123I]β-CIT, [123I]FP-CIT and other dopamine transporter ligands

Possibilities to determine the integrity of the dopaminergic system in vivo emerged by the finding that cocaine analogues are very selective and efficient ligands for the so-called dopamine transporters, which are expressed in the striatal dopaminergic projections182. Thus, it was proposed that labeling of the dopamine transporter in vivo with ligands that can be visualized by brain imaging techniques could provide a valuable tool to investigate the extent of degeneration of the dopaminergic system in Parkinson’s disease. Indeed, single photon emission computed tomography (SPECT) with the [123I]-labeled cocaine analog 2-β-carbomethoxy-3-β-(4-iodophenyl)-tropane ([123I]β-CIT) revealed a dramatic loss of striatal dopamine transporters in Parkin-son’s disease patients52,53. Likewise, SPECT studies with other cocaine analogs such as [123I]2-β-carbomethoxy-3-β-(4-fluorophenyl)-N-(1-iodoprop-1-en-3-yl)nortropane (altropane183) and the [123I]fluoropropyl derivative of β-CIT ([123I]FP-CIT36,38) demon-strated similar losses in transporter binding as with β-CIT. An important difference between altropane and FP-CIT, on the one hand, and β-CIT, on the other, is the much slower kinetics of the latter one. For instance, specific FP-CIT accumulation can reli-ably be measured 3 h following injection, whereas 24 h is needed to require the optimal specific/nonspecific binding ratio following the injection of β-CIT.

3.2. Monitoring the degeneration rate of dopaminergic neurons in Parkinson’s disease with dopamine transporter ligands for SPECT

In light of its possible application in longitudinal studies in Parkinson’s disease pa-tients as a device to reliably monitor both the progression of the degenerative process and the effect(s) of putative neuroprotective agents, the question arises whether the [123I]β-CIT SPECT procedure is sensitive enough to discriminate between early and late stage Parkinson’s disease. Vermeulen et al.54 investigated [123I]β-CIT binding in healthy

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controls, early stage (untreated, disease history <2.5 years) Parkinson’s disease patients and Parkinson’s disease patients with a long history (>10 years) and treated with dopa-minomimetics (late stage Parkinson’s disease). No differences could be detected between caudate nucleus [123I]β-CIT binding in controls and early stage Parkinson’s disease pa-tients (94% of control), whereas [123I]β-CIT binding in the caudate nucleus of late stage Parkinson’s disease patients (35% of control) differed statistically significant from that of control and early stage Parkinson’s disease patients. [123I]β-CIT binding in the putamen was significantly decreased in both the early (52% of control) and late stage Parkinson’s disease patients (20% of control), the binding of [123I]β-CIT in the putamen being sig-nificantly more decreased in late than in early stage Parkinson’s disease patients. In both early and late stage Parkinson’s disease patients binding of the ligand was more reduced in the putamen than in the caudate nucleus. This differential loss of [123I]β-CIT binding in putamen and caudate nucleus is in agreement with results from autopsy studies that revealed 80% decrease of dopamine concentration in the putamen and 40% decrease of dopamine level in the caudate nucleus151,184 and confirms other in vivo findings of Innis et al.50. The fact that this differential loss can be demonstrated in vivo testifies to the power of this brain imaging method. Altogether, these results indicated that the [123I]β-CIT SPECT procedure is indeed sensitive enough to discriminate Parkinson’s disease patients in the early stage from those in the late stage of the disease. In a study performed by our group, the annual rate of decline in dopamine transporters in early stage Parkinson’s disease was investigated by making two consecutive scans, 12 months apart, with [123I]β-CIT and [123I]FP-CIT as ligands185. Forty six and twenty de novo pa-tients were examined on two occasions with [123I]β-CIT and [123I]FP-CIT, respectively. The first SPECT-scan was made immediately after patients entered the study, whereas the second scan was made 12 months thereafter. The results showed a statistically signif-icant decrease of 7 and 8% in binding ratios in the striatum for [123I]β-CIT and [123I]FP-CIT, respectively. This is a dramatic decrease in binding ratios as compared to healthy individuals in which the reported decrease amounts to less than 1% per year186,187. Thus, using [123I]β-CIT – or [123I]FP-CIT SPECT, one might not only be able to monitor the rate of progression of the desease process in Parkinson’s disease, but, more importantly, also to estimate in a quantitative manner the effect of neuroprotective agents on the disease process within a period of 1 year in a relatively small group of patients.

3.3. PET studies with [18F]DOPA and other ligands

Besides SPECT investigations with dopamine transporter ligands, brain imaging stud-ies of the dopaminergic deficit in Parkinson’s disease in vivo have been performed us-ing [18F]DOPA positron emission tomography (PET) scanning188. The application of this PET-ligand is based on the assumption that [18F]DOPA selectively accumulates in dopa-minergic terminals in the striatal (sub)regions. However, in the early phase of Parkinson’s disease, this technique reveals a less dramatic decrease in accumulation of [18F]DOPA

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as compared to the decrease in transporter binding found with SPECT. For instance, in a series of 27 patients with early stage Parkinson’s disease (Hoehn and Yahr staging scale 1.8), Morrish et al.189 found an average [18F]DOPA accumulation of 62% of control in the putamen with PET, whereas Tissingh et al.36 demonstrated in a similar group of 21 patients (Hoehn and Yahr staging scale 1.8) 43% binding in the putamen by using [123I]FP-CIT SPECT. Moreover, in a group of 33 early stage Parkinson’s disease patients (Hoehn and Yahr staging scale 1.7), these same authors found less than 35% binding in the putamen when using [123I]β-CIT SPECT190. From these data, it might be concluded that dopamine transporter ligands are better compounds for the early diagnosis of Parkinson’s disease than fluoro-DOPA. This hypothesis has been underscored by a very elegant study performed recently by Itoh et al.191 in rats. In this study, an early to advanced stage model of Parkinson’s disease was mimicked by injecting 1–10 μg of 6-hydroxydopamine into the substantia nigra. Using adjacent sections of the same animals, the binding of [125I]β-CIT and the uptake of [14C]L -DOPA were evaluated in the striatum 4 weeks after induction of the lesion. The results of this investigation showed a decrease in both [125I]β-CIT bind-ing and [14C]L-DOPA uptake, in parallel with a decrease in dopaminergic neurons from early to advanced stage models, the decrease in L-DOPA uptake being always smaller than that of β-CIT. Thus, L-DOPA accumulation apparently underestimates the decrease in dopaminergic neurons, which indeed supports the notion that transporter ligands are better suited to establish an early diagnosis of Parkinson’s disease. Dopamine transporter ligands have been used also for PET. For instance, the SPECT ligand FP-CIT can also be applied for PET studies, just by replacing the fluorine atom by a 18F isotope. Studies with [18F]FP-CIT demonstrated an age-related decline in dopamine transporter binding in normal subjects (approximately 7% per decade) as well as a significant reduction in patients with idiopathic Parkinson’s disease, which correlated with disease severity187. Similar results have been reported by using [11C]dihydrotetrabenazine as a ligand for the monoaminergic storage vesicles192.

3.4. Monitoring the degeneration rate of dopaminergic neurons in Parkinson’s disease with [18F]DOPA PET

A few studies have investigated the rate of progression of the loss of striatal [18F]DOPA metabolism in patients wit Parkinson’s disease. In a group of 32 patients, Morrish et al.193 made two consecutive PET scans 18 months apart. The mean annual rate of deteriora-tion in [18F]DOPA accumulation varied according to striatal region, with the putamen showing the highest mean rate of progression, i.e., 9% of the initial value. This outcome compares well with a previous study from the same group194 in which a mean annual rate of reduction in [18F]DOPA accumulation in the putamen of 12.5% per annum was obtained. Interestingly, these percentages are in close agreement with the annual de-cline reported in the aforementioned SPECT study with [123I]β-CIT and [123I]FP-CIT as ligands in early-stage Parkinson’s disease185, but are in sharp contrast to a study per-

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5�

formed by Vingerhoets et al.195. These authors performed [18F]DOPA PET scans on two occasions in a group of 16 patients with idiopathic Parkinson’s disease 7 years apart and found an annual decline in [18F]DOPA accumulation of 1.7% in patients vs. 0.3% in controls. However, from this study, it is not clear of course as to whether the percentage decline during the first year differs from that in later years, in other words, whether the progression proceeds in a linear or in an exponential way. In conclusion, it has been clearly demonstrated that both PET and SPECT are valuable methods for the early di-agnosis of Parkinson’s disease. Additionally, although yet to be established definitely, both brain imaging techniques appear to offer the possibility to estimate both the rate of degeneration of the dopaminergic system in Parkinson’s disease and to assess the ef-fectiveness of (putative) neuroprotective agents.

CHAPTER 4.1

Dopamine transporter SPECT is a useful method to monitor

the rate of dopaminergic degeneration in early-stage Parkinson´s disease

Adapted from: A Winogrodzka, P Bergmans, J Booij,

EA van Royen, AGM Janssen, ECh Wolters

Journal of Neural Transmission 2001;108:1011–1019

and A Winogrodzka, P Bergmans, J Booij, EA van Royen, JC Stoof, ECh Wolters

Journal of Neurology, Neurosurgery and Psychiatry 2003;74:294–298

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Summary

Objective: To examine the validity of [123I]β-CIT and [123I]FP-CIT SPECT for monitor-ing the progression of dopaminergic degeneration in Parkinson’s disease; to investigate the influence of short term treatment with D2 receptor agonists on striatal [123I]β-CIT binding; and to determine the sample size and frequency of SPECT imaging required to demonstrate a significant effect of a putative neuroprotective agent.Methods: Two groups of early stage Parkinson’s disease patients were examined twice, a mean of 12 months apart: group 1 with 50 patients examined with [123I]β-CIT and group 2 with 20 patients examined with [123I]FP-CIT. The mean annual change in the ratio of specific to nonspecific [123I]β-CIT and [123I]FP-CIT binding to the striatum was used as the outcome measure. Results: The average annual decrease in striatal [123I]β-CIT and [123I]FP-CIT binding ratios was found to be about 8% (of the baseline mean). Comparisons of scans done in nine patients under two different conditions – in the off state and while on drug treat-ment with D2 receptor agonists– showed no significant alterations in the expression of striatal dopamine transporters as measured using [123I]β-CIT SPECT. Power analysis indicated that to detect a significant (p<0.05) effect of a neuroprotective agent with 0.80 power and 50% of predicted protection within 2 years, 85 patients are required in each group, when the effects are measured by means of changes in [123I]β-CIT binding ratios in whole striatum.Conclusions: Our findings indicate that SPECT with both [123I]β-CIT and [123I]FP-CIT seems to be a useful tool to investigate the progression of dopaminergic degeneration in Parkinson’s disease and may provide an objective method of measuring the effec-tiveness of neuroprotective therapies. Short term treatment with a D2 agonist does not have a significant influence on [123I]β-CIT binding to dopamine transporters. If the lat-ter finding is replicated in larger groups of patients, and using [123I]FP-CIT as well, it supports the suitability of these techniques for examining the progression of neurode-generation in patients being treated with D2 receptor agonists.

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Dopamine transporter SPECT is a useful method to monitor the rate of dopaminergic degeneration in early-stage Parkinson´s disease

Introduction

The pathophysiological hallmark of Parkinson’s disease (PD) is a slow, progressive de-generation of dopaminergic neurons in the substantia nigra72. Standard therapeutic interventions are aimed at replenishment of empty dopamine stores with levodopa or substitution with dopamine receptor agonists. However, in the long term this symptom-atic therapy fails. Currently, various neuroprotective agents are being developed, with the intention of treating the cause of the disease and thereby delaying the degeneration of dopaminergic neurons. To evaluate the effectiveness of such neuroprotective agents, it is critical, however, to develop methods that can reliably measure progression of dopami-nergic degeneration. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging could provide objective tools for measuring the effectiveness of putative neuroprotective agents and monitoring disease progression.

In several studies it has been shown, by using [18F]dopa PET, that the presynap-tic dopaminergic degeneration in PD is faster than in normal aging-193-195. SPECT with [123I]β-CIT SPECT and [123I]FP-CIT has been used to investigate the presynaptic dopa-minergic system in PD, by assessing the concentration of striatal dopamine transport-ers. A possible disadvantage of using [123I]β-CIT as SPECT radioligand for dopamine transporter, is the fact that a stable level of radioactivity in the striatum is only reached between 20 and 30 h post-injection. This indicates that the optimal image acquisition can be performed only the day after the injection of [123I]β-CIT which makes it less conve-nient for routine use. The faster kinetics of [123I]FP-CIT allow adequate image acquisition as early as 3 h post-injection. Several studies have shown that striatal binding of both, [123I]β-CIT and [123I]FP-CIT in PD patients was decreased compared to healthy con-trols and correlated well with symptom severity36-39,41. SPECT with both [123I]β-CIT and [123I]FP-CIT is considered a highly reproducible technique, which could be of value in monitoring the progression of dopaminergic degeneration in PD185,196..

Our aim in this study was to investigate whether serial SPECT imaging with [123I]β-CIT and [123I]FP-CIT can be used as a marker of PD progression. We undertook two imaging series with both radioligands in two groups of early stage PD patients: group 1 with 50 PD patients examined with [123I]β-CIT SPECT and group 2 with 20 PD patients examined with [123I]FP-CIT SPECT. We also estimated the sample size and imaging in-terval necessary to predict the effectiveness of a putative neuroprotective agent.

Methods

Subjects

For the “progression study”, a group of 50 PD patients (group A; 31 men, 19 women) was examined with [123I]β-CIT SPECT and another group of 20 patients (group B; 16 men, 4 women) was examined with [123I]FP-CIT SPECT. The patients were recruit-

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ed from the movement disorders unit of our outpatient clinic. The diagnosis of PD was established according to the UK Parkinson’s disease Society Brain Bank Criteria61. The mean (SD) age of the patients at the time of the first imaging was in the group A 56.7 (9.3) years, range 37 to 71, and in the group B 55.4 (9.9) years, range 43–73. The mean duration of PD was 2.7 (2.2) years in the group A, and 2.5 (1.3) years in the group B. The Hoehn and Yahr Staging Scale90 and the Unified Parkinson’s Disease Rating Scale (UPDRS89) were used to assess the stage and severity of the disease at the time of first imaging. A detailed clinical and demographical description of the patients is given in Table 1. Each patient was imaged on two occasions, with a mean scan-to-scan interval of 51 (7) weeks in group A, and 48 (3) weeks in group B. Imaging was always performed on the same equipment and following the same protocol. All patients gave written informed consent to the research protocol, which was approved by the medical ethics committee of the hospital.

Drug treatment

All patients in the “progression study” were drug-naive at the time of the first scan, after which dopaminergic treatment (levodopa or D2 receptor agonist) was initiated in all patients in group A and in 15 patients in group B; 5 patients in group B were still drug-naive at the time of the second SPECT image. A previous study in patients with PD showed no significant effects of subchronic administration of levodopa or L-sele-giline on striatal [123I]β-CIT binding to dopamine transporter197. Likewise, D2 receptor agonists appear to have no influence on the expression of the striatal dopamine trans-porter198-200, as showed in animal studies. However, the effects of these drugs on striatal [123I]β-CIT binding in PD patients have only been examined in one study201.

Table 1. Clinical and demographical data of PD patients (group A; n=50; examined with [123I]β-CIT and group B; n=20 examined with [123I]FP-CIT ) during first imaging session

Group A

n=50

Group B

n=20

Male/ female 31/19 16/4

Age (years) 56.7(9.3) 55.4 (9.9)

PD duration (years) 2.7 (2.2) 2.5 (1.3)

Hoehn and Yahr stage 1.9 (0.6) 1.6 (0.6)

UPDRS motor score 19.2 (6.7)) 16.5 (6.9)

Data are presented as mean (SD)Abbreviations: PD = Parkinson’s disease; UPDRS = Unified Parkinson’s Disease Rating Scale.

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Effect of D2 receptor agonist on striatal [123I]β-CIT binding

In order to assess the effects of D2 receptor agonists on striatal [123I]β-CIT binding in our study, nine PD patients (seven men, two women; age 61.5 (8.2) years; PD duration 3.2 (1.5) years) were imaged twice: first, when not previously exposed to D2 receptor agonist treatment or when withdrawn from it, and second, while on treatment with D2 receptor agonist. The scan to scan interval was two to five weeks. Six of the patients had not previously received D2 receptor agonist treatment at the time of the first imag-ing. In these six patients (protocol 1), treatment with a D2 receptor agonist (pergolide or pramipexol) was started one day after the baseline imaging, following an increasing schedule up to 2.0 mg/day for pergolide and 2.25 mg/day for pramipexol (table 2). In these patients, the second imaging was done after four to five weeks while they were on treatment with the D2 receptor agonist. In the last week before the second imaging, the daily dose of the D2 receptor agonist was maintained stable.

The other three patients in the D2 receptor agonist assessment study were first imaged while on a stable dose of pergolide, which has been given as monotherapy for about a year. After the initial imaging, the drug was withdrawn for two to three weeks and the second imaging was done (protocol 2).

The effects of D2 receptor agonist treatment on [123I]β-CIT binding measures and the treatment details of the patients are summarised in table 2.

Table 2. Effects of treatment with D2 agonists on ratios of specific to non-specific [123I]β-CIT binding in the whole striatum.

No Sex Protocol Drug (mg/day)Striatal [123I]β-CIT binding ratio

on medication drug naive or withdrawn

1 F 2 Pergolide 1.0 1.72 1.79

2 M 2 Pergolide 2.0 1.95 2.30

3 M 2 Pergolide 2.0 1.63 1.66

4 M 1 Pergolide 2.0 2.13 1.88

5 F 1 Pramipexol 2.25 2.86 2.49

6 M 1 Pergolide 1.5 1.86 2.10

7 M 1 Pergolide 1.5 3.25 3.78

8 M 1 Pergolide 2.0 2.22 2.22

9 M 1 Pergolide 1.0 2.70 2.90

Mean (SD) 2.26 (0.6) 2.35 (0.7)

Abbreviations: M = male; F = female; Protocol 1 = drug naive at the time of first imaging; Protocol 2 = approximately 1 year on monotherapy at the time of first imaging.

Chapter 4.1

64

SPECT camera

The Strichman Medical Equipment 810X system was used for SPECT imaging. The sys-tem is equipped with 12 individual crystals, each with a focussing collimator. The transaxial resolution of this camera is 7.6 mm full width at half maximum of a line source in air. The energy window was set at 135–190 keV. Data acquisition took place in a 128×128 matrix.

SPECT imaging

All patients received potassium iodide orally in order to block thyroid uptake of free radioactive iodide. [123I]β-CIT and [123I]FP-CIT (specific activity >185 MBq/nmol; radiochemical purity >99%) were injected intravenously at an approximate dose of 110 MBq. 123I labeling, acquisition, attenuation correction and reconstruction of images was done as described before36,38,42,196,202. The measured concentration of radioactivity was expressed as Strichman Medical Units (SMUs; 1 SMU = 100 Bq/ml). Image acquisi-tion was always started at 24 hr after injection of [123I]β-CIT and 3 hr after injection of [123I]FP-CIT. Slices were acquired during 300 s periods, after positioning of the patient’s head in the camera, with beams from gantry-mounted lasers oriented parallel to the canthomeatal line (CM line), from the CM line to the vertex using a interslice distance of 10 mm. Imaging was always done using the same equipment and following the same protocol. During the second image session, all efforts were made to ensure that the patient’s head in the camera conformed to the position used during the first image ses-sion. To achieve this, the distances from the meatuses of the ears and from the orbital angles to the position of the laser beams were recorded. In previous studies we showed that this procedure is highly reproducible196.

Data processing

For analysis of striatal [123I]β-CIT and [123I]FP-CIT binding, two transversal slices rep-resenting the most intense striatal binding were summed. A standard region of interest (ROI) template, constructed according to a stereotactic atlas including fixed regions for whole striatum and occipital cortex, was placed bilaterally on the combined image, as described previously36. Estimates of specific striatal binding were made by subtracting occipital counts (non-specific binding) from striatal counts. The ratio of specific to non-specific striatal [123I]β-CIT and [123I]FP-CIT binding was then calculated by dividing the specific striatal uptake by the occipital uptake63.

65


Statistics

The relation between the initial radioligand binding and UPDRS was measured using the Spearman rank correlation. A paired samples two tailed t test (for group A) and Wilcoxon Signed Ranks Test (for group B) were used to examine the change between baseline and follow-up imaging results; Wilcoxon Signed Ranks Test was used to com-pare the change in [123I]FP-CIT binding ratio in medicated and non-medicated patients in group B. The mean annual rate of decline in radioligand binding ratios was expressed as a percentage of the baseline image and was calculated for each patient using the fol-lowing formula:

{[follow up SPECT imaging – baseline SPECT imaging]} over {[baseline SPECTimaging]} times 100"%"

[ follow up SPECT imaging – baseline SPECT imaging ]

[baseline SPECT imaging ]×100%

For the assessment of the effects of D2 receptor agonists on [123I]β-CIT binding, the two conditions (on drug treatment, and either withdrawn from drug treatment or drug-naive) were compared using the Wilcoxon signed ranks test.

Power analysis was performed in order to estimate the sample size and the scan in-terval required to demonstrate a significant neuroprotective effect of agents with various degrees of predicted protection. The analysis was performed assuming the annual rate of dopaminergic degeneration based on the data obtained in the present study with [123I]β-CIT. The standard deviation of the mean annual change in [123I]β-CIT binding was used as a measure of variance. Sample size was determined using Altman’s normogram203.

In case of multiple comparisons the Bonferroni correction was used. Significance was assumed at a probability (p) value of <0.05.

Results

No significant difference in age, disease severity, or radioligand binding measures was found between male and female subjects.

Progression of dopaminergic degeneration

Hoehn and Yahr stage of the patients at the time of the first image was signifi-cantly correlated with baseline [123I]FP-CIT binding ratios in the regions of interest (Spearman rank correlation; correlation coefficients were –0.66 (p<0.002) and –0.62 (p<0.003) for the ipsilateral and contralateral striatum, respectively. The baseline ratios of specific to non-specific [123I]β-CIT binding in all striatal regions of interest showed significant correlations with baseline motor UPDRS score (r values varying from -0.32 to -0.5; p<0.02; fig 1)

Chapter 4.1

66

A significant decrease in specific to non-specific radioligand binding ratios be-tween the two consecutive scans was found in all regions of interest, for both [123I]β-CIT and [123I]FP-CIT (table 3). The relative annual rate of decrease in [123I]β-CIT and [123I]FP-CIT binding ratios was about 8% of the baseline value in striatal regions.

No correlation was found between the rate of progression in the regions of interest and the baseline binding ratios, the duration of the PD symptoms, nor the severity of the disease expressed by means of UPDRS motor score.

Figure 1. Correlation (Spearman rank) of the unified Parkinson’s disease rating scale (UPDRS) motor score with specific to non-specific [123I]β-CIT binding in striatum, putamen, and caudate nucleus in 50 patients with Parkinson’s disease.

Striatum

r=-.51; p<.001

UPDRS motor score

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Caudate nucleus

r=-.52; p<.001

UPDRS motor score

403020100

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Putamen

r=-.46; p<.001

UPDRS motor score

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Table 3. The mean annual change in the ratio of specific to nonspecific [123I]β-CIT and [123I]FP-CIT SPECT binding

Meanimage 1

Meanimage 2

Mean relativechange (%)

Striatum whole

[123I]FP-CIT[123I]β-CIT

1.27 (0.3)2.60 (0.7)

1.15 (0.3)2.40 (0.7)

-8.08 (12.2)*-7.60 (17.9)*

Striatum ipsi


1.45 (0.4)2.90 (0.8)

1.31 (0.3)2.60 (0.8)

-8.09 (13.9)*-7.50 (17.6)*

Striatum contra


1.09 (0.3)2.30 (0.6)

0.99 (0.3)2.10 (0.7)

-8.35 (12.7)*-7.10 (20.7)*

Values are mean (SD)Abbreviations: ipsi = ipsilateral; contra = contralateral (side opposite that of initial presentation of motor signs)*Significant relative difference between the mean values of two imaging assessment.

67


Effect of D2 receptor agonist on striatal radioligand binding

Treatment with D2 receptor agonists in the group A of PD patients (examined with [123I]β-CIT) did not cause significant changes in striatal [123I]β-CIT binding ratios, as measured by serial SPECT imaging under two different conditions (protocol 1 and protocol 2) described above: on treatment versus drug naive or withdrawn from drugs (2.26 (0.6) versus 2.35 (0.67); p=0.4; table 2).

In group B of PD patients (examined with [123I]FP-CIT), no statistically signifi-cant difference was found in the annual rate of decrease in [123I]FP-CIT binding ratios between medicated and non-medicated PD patients.

Power analysis

Power analysis indicated that in order to detect a significant effect of a neuroprotec-tive agent with 0.80 power and 50% of predicted protection within 2 years, 85 patients would be required in each group, when the effects are measured by means of changes in [123I]β-CIT binding ratios in the whole striatum. For a trial with an agent with 30% of predicted neuroprotection 238 patients in each group would be required. Assuming linear decline of dopaminergic function, it was calculated that extension of the scan-ning interval to 5 years reduces the required sample size to 14 patients in each group, for a trial with an agent with 50% of predicted protection, and 38 patients in each group, for a trial with 30% of predicted protection (table 4).

Discussion

In our study we investigated the value of SPECT with [123I]β-CIT and [123I]FP-CIT in monitoring the progression of PD by serial evaluation in a group of early-stage PD pa-tients204-206. [123I]β-CIT SPECT has been shown to detect loss of DA transporters compa-rably to [123I]FP-CIT SPECT207.

Table 4. Indication of the sample size and scan-to-scan interval ([123I]β-CIT SPECT) required to give 80% power of detecting a significant (p<0.05) effect of a neuroprotective agent, with predicted protection of 50% and 30% (assuming the reported relative rate of dopaminergic degeneration).

Time interval

1 year 2 years 5 yearsprotection protection protection

50% 30% 50% 30% 50% 30%

Striatum (whole)

Ipsi

Contra

340

346

534

949

961

1483

85

87

134

238

241

371

14

14

22

38

39

60

Abbreviations: ROI = region of interest; ipsi = ipsilateral; contra = contralateral (side opposite that of initial presentation of motor signs).

Chapter 4.1

6�

The radioligand binding ratios obtained at the first series of imaging were in PD patients significantly lower than in control subjects36,190. Moreover, a significant corre-lation was found between baseline radioligand ([123I]β-CIT and [123I]FP-CIT) binding ratios and the disease severity in drug-naive patients. These results are in line with the findings of previous studies36-39, indicating that SPECT with [123I]β-CIT and [123I]FP-CIT is a sensitive marker of disease severity in PD.

The test/retest reproducibility of both [123I]β-CIT and [123I]FP-CIT SPECT has re-cently been investigated by Seibyl et al40 and Booij et al196. These investigators showed that SPECT imaging with [123I]β-CIT and [123I]FP-CIT is a highly reproducible measure of striatal dopamine transporters in PD patients and suggested the suitability of this method in the serial evaluation of dopaminergic degeneration in PD.

In the present study, we showed that the mean annual rate of dopaminergic de-generation in striatum reached statistical significance at about 8%. These findings fit well with the results of other SPECT studies190,205,206,208,209, successfully demonstrating the applicability of dopamine transporter imaging with both, [123I]β-CIT and [123I]FP-CIT to assess disease progression in PD. Interestingly, also a PET study, using [18F]CFT as a radiotracer for the dopamine transporter, also showed that the rate of annual decline in early PD patients, was 13,1% and 12,5% in the putamen and caudate nucleus, re-spectively210. In addition, several [18F]DOPA PET studies showed progression of disease in PD193,195. For example, Morrish et al193 reported mean annual rates of dopaminergic degeneration in PD of 9%.

Van Dyck et al investigated age related changes in dopamine transporter binding with [123I]β-CIT SPECT in human controls186 and found approximately an 8% decline per decade. In this context, our results show that the rate of progression of dopaminer-gic degeneration is much faster in PD than in normal ageing.

Though the most of the patients in the present study were under dopaminergic drug treatment (levodopa or a D2 agonist) at the time of the second imaging, Laruelle et al showed that infusion of high dose levodopa failed to displace striatal [123I]β-CIT binding in non-human primates211. In line with this observation, Innis et al demonstrat-ed recently that treatment with levodopa or L-selegiline in PD causes neither significant occupancy nor modulation in the number of striatal dopamine transporters labeled with [123I]β-CIT197. Moreover, Ahlskog and coworkers201 reported that short term treat-ment with the dopamine D2 agonist pergolide did not significantly influence binding of [123I]β-CIT to dopamine transporters in PD.

In order to assess the effects of D2 agonists on [123I]β-CIT binding to the dopamine transporters we also undertook sequential imaging in nine PD patients under two differ-ent conditions: on D2 agonist treatment, and drug-naive or withdrawn from D2 agonist treatment. Our results show that short term treatment with D2 agonists did not cause any significant changes in the binding of [123I]β-CIT to striatal dopamine transporters. This finding confirms the results of previous animal experiments investigating the influence of several dopamine receptor agonists on the striatal dopamine transporter198-200. Finally,

6�


in the present study, in the group of PD patients examined with [123I]FP-CIT, the rate of decrease in binding after 1 year was comparable between medicated and non-medicated PD patients, which also may indicate no significant effects of dopaminergic medication. Therefore, measurement of dopaminergic degeneration in the present study was based on the assumption that treatment with D2 agonists or levodopa has no significant influ-ence on radioligand binding to striatal DA transporters. Nonetheless, all of these stud-ies, including the present one, are limited by small sample sizes (and consequently by limited power), as well as by short duration, and therefore do not exclude the possibility that pharmacological effects could emerge in larger or longer term studies.

Several techniques are now available for imaging nigrostriatal dopaminergic neu-rons (for a review, see Booij and colleagues212). In the present study, we used two radio-tracers which bind to dopamine transporter. This transporter is located exclusively on nerve terminals of dopaminergic cells. Several studies have shown that the loss of striatal dopamine transporters in PD is in concordance with the loss of nigrostriatal dopamine neurones182. In addition, necropsy studies in PD patients have shown that measurements of dopamine transporter density are highly correlated with striatal dopamine levels213. Experimental studies in rats and monkeys have also validated the fact that drug induced losses of nigrostriatal cells are correlated with loss of dopamine transporters, as mea-sured by [123I]/[125I]β-CIT191,214. Moreover, several SPECT studies with [123I]β-CIT and [123I]FP-CIT have shown loss of dopamine transporters in PD, and correlations between motor signs and severity of loss of these transporters36-39. These studies have shown the validity of using dopamine transporter imaging to assess the integrity of presynaptic do-paminergic nerve terminals in PD. In fact SPECT with [123I]β-CIT and [123I]FP-CIT as-sesses the expression of dopamine transporters on surviving dopaminergic cells. An im-portant study of Lee et al suggested that the dopamine transporter is downregulated on surviving dopaminergic neurons in PD215. This observation is in line with experimental studies suggesting that the loss of dopaminergic neurones is functionally compensated by an increase in synthesis and release of dopamine from surviving dopamine nerve terminals, as well as by a reduced rate of dopamine inactivation in presynaptic dopamine nerve terminals216. In the light of all these data, we cannot exclude the possibility in the present study that the loss of dopamine transporters in PD results from a combination of cell loss (and consequently loss of dopamine transporters) and downregulation of do-pamine transporters on surviving neurons. However, if the loss of radioligand binding to dopamine transporter in PD can be explained partly by downregulation, it seems un-likely that this regulation will disappear, whereas degeneration of dopaminergic neurons will progress. Consequently, the possible occurrence of downregulation on surviving dopaminergic nerve terminals in PD may not hamper the applicability of SPECT tech-nique with [123I]β-CIT and [123I]FP-CIT in monitoring disease progression.

Our study allows sample size calculations for future studies on the evaluation of neuroprotective treatments (table 4). We estimated that, in order to show a signifi-cant effect of a neuroprotective agent with 80% power and 50% of predicted protection

Chapter 4.1

70

within 2 years, 85 patients are required in each group, when the effects are measured in the whole striatum. For a trial with an agent with 30% of predicted protection 238 patients are required. As expected, extending the scanning interval to 5 years reduces the required sample size remarkably. All estimations are based on the assumption of a constant rate of dopaminergic degeneration through the initial course of the disease. The linear evolution seems a reasonable approximation for the relatively brief period of time involved in the present study. However, if the degeneration follows an exponential course, slowing down in the later disease stages (a reasonable hypothesis, suggested in several studies194,209), then a larger sample size would be required to detect the same neuroprotective effect.

Conclusions

Our study shows that SPECT with [123I]β-CIT and [123I]FP-CIT allows to measure the rate of dopaminergic degeneration in early-stage Parkinson’s disease. Our observations, in combination with the fact of wide availability of the SPECT technique and, in case of [123I]FP-CIT, also an advantage of image acquisition 3 h post-injection, makes it a good alternative for [18F]dopa PET as a method for estimating the effectiveness of putative neuroprotective therapies in large clinical trials. Our study also showed that short term treatment with D2 agonists did not influence [123I]β-CIT binding to dopamine trans-porters in a small group of patients. If replicated in larger groups of patients, this finding confirms the applicability of [123I]β-CIT for examining the progression of dopaminergic degeneration in PD patients who are on these drugs.

CHAPTER 4.2

Disease-related and drug-induced changes in dopamine transporter

expression might undermine the reliability of imaging studies

of disease progression in Parkinson’s disease

A Winogrodzka, J Booij, ECh Wolters

Parkinsonism and Related Disorders, 2005;11(8):475–84

Chapter 4.2

72

Summary

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Standard ther-apeutic interventions are aimed at replenishment of empty dopamine stores with le-vodopa or substitution with dopamine receptor agonists. However, in the long term this symptomatic therapy fails. Currently, various neuroprotective agents are being de-veloped, with the intention to slow down the degeneration of dopaminergic neurons. In this context, the early identification of persons at risk to develop the disease as well as the assessment of the effectiveness of putative neuroprotective agents, are critical is-sues. Dopamine transporter (DAT) scintigraphy with single photon emission computed tomography (SPECT) has been used to assess the dopaminergic function in PD. Initial studies with several radioligands show significant loss of DAT binding in PD patients as compared to controls. In this paper we review the evidence on the utility of DAT imag-ing with SPECT in early PD detection as well as in monitoring neurprotection.

73

Disease-related and drug-induced changes in dopamine transporter expression might undermine the reliability of imaging studies of disease progression in Parkinson’s disease

Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder characterized pathological-ly by slow, but progressive, loss of dopaminergic neurons in the substantia nigra and ventral tegmental area. The biochemical consequence is a marked reduction of striatal dopamine concentration combined with a varying degree of deterioration of the cho-linergic, serotonergic, and noradrenergic system. The clinical consequence is a variety of severe and progressive motor and non-motor symptoms. Based on recent evidence, it has been accepted that there is a preclinical/premotor phase in PD in which motor be-haviour in patients is still unimpaired despite a substantial loss of dopamine containing neurons217. When patients become symptomatic and fulfill the clinical criteria for PD, approximately 60% of dopaminergic neurons are lost36,42. In this “early” clinical stage the standard therapeutic interventions are aimed to replenish empty dopamine stores with levodopa or substitute for dopamine with dopamine receptor agonists. However, on the long term, this symptomatic therapy fails and progressive neuronal multisystem degen-eration results in a severe functional disability. The existence of preclinical stage in PD offers the opportunity for presymptomatic diagnosis and treatment. Indeed, converting the symptomatic therapy into disease-modifying therapy has become a major subject of research in PD. Currently, various neuroprotective agents are being developed, with the intention to delay the progression of degeneration of dopaminergic neurons. A recently performed systematic review of candidate drugs as putative neuroprotective agents for trials in PD identified 12 potential neuroprotective compounds26. Since even the earliest phase of PD is characterized by severe loss of dopaminergic neurons, protective therapy should be offered to subjects who are at risk to develop the disease or at least it should be initiated during the preclinical phase of the disease, before the symptoms occur. There-fore, early identification of persons at risk may be essential for successful neuropro-tection. To evaluate the effectiveness of neuroprotective agents, it is critical to develop methods that can accurately measure (progression) of dopaminergic degeneration.

During the past decade functional neuroimaging of the dopaminergic system using single photon emission computed tomography (SPECT) or positron emission tomography (PET) became a validated and well accepted tool to assess the integrity of nigrostriatal dopaminergic nerve terminals in vivo. The two most commonly used imaging modalities are 18F-Dopa PET, primarily reflecting dopamine synthesis (decar-boxylase activity) and storage, and dopamine transporter (DAT) scintigraphy with PET or SPECT using various radioligands. In this review we will mainly focus on DAT im-aging. DAT is a protein exclusively located on presynaptic nigrostriatal terminals of dopamine containing neurons and serves as a marker for the integrity of the presynaptic dopaminergic innervation218. Several PET and SPECT radioligands for DAT imaging have been synthesized and evaluated successfully, such as [11C]nomifensine, [11C] or [18F]β-CFT, [11C]RTI-32, [11C] or [123I]PE21, [99mTc]TRODAT, [123I]IPT. The most widely used radiotracers are [123I]β-CIT and [123I]FP-CIT. Initial studies with these techniques

Chapter 4.2

74

have shown significant loss of DAT in PD patients as compared to controls36. In several cross-sectional studies correlations were found between striatal [123I]β-CIT or [123I]FP-CIT binding and PD symptom severity37-39. DAT scintigraphy turned out to be sensitive to the loss of dopamine transporters in the very early stages of PD37,38,219, and even before the onset of motor signs27,42,43.

The possibility to measure dopaminergic function in PD using DAT imaging, as well as the observed correlations between in-vivo DAT measures and disease severity has led to the initiation of studies on PD progression. Longitudinal studies have shown the annual rate of reduction of striatal DAT uptake to be about 5-13% in PD patients. These results are in line with the rates of binding decline with 18F-dopa PET suggesting that DAT imaging can be used for monitoring PD progression. Based on the results of the progression studies, the rate of decline in DAT radioligand binding was suggested to be used as primary endpoint for testing the possible neuroprotective effects of drugs. The use of imaging techniques in this context would be especially attractive since the clinical endpoints used to evaluate the effect of putative neuroprotectants on disease progression in PD are easily confounded by any symptomatic benefit of the drug. The debate raised, however, whether those methodologies are able to prove neuroprotective properties of drugs that are being tested, and, especially, whether chronic treatment with dopaminergic drugs may influence the markers that are being measured220-222.

In this paper we review the evidence on the utility of DAT imaging in early PD detection, measuring PD progression as well as monitoring neuroprotection, critically addressing the issues of sensitivity and reproducibility as well as potential regulatory effects of drugs on DAT imaging results.

Early PD detection

In the era of emerging neuroprotective possibilities early disease detection becomes crucial as it offers the promise of disease modifying treatment in early or even preclini-cal stage of the disease. Several tools can be considered to be used complementarily to identify the subjects at risk during very early/premotor PD stage. The identification of specific gen mutations can be used as a genetic marker for early PD detection in families with non-sporadic PD. Subtle visuomotor control abnormalities and subtle neurocognitive dysfunction appear to precede early motor PD symptoms and might be useful as clinical markers of early disease223,224.

Loss of olfaction has been found in asymptomatic relatives of PD patients30 as well as in the earliest clinical stage of PD29 indicating a potential value of hyposmia as a clinical marker for early PD detection. There is evidence suggesting that func-tional imaging of the dopaminergic system with PET and SPECT may also provide a marker of preclinical dopaminergic degeneration. The SPECT DAT imaging studies in early PD patients with strictly unilateral disease showed a bilateral loss of striatal

75


dopamine transporters suggesting the possibility to identify subjects in preclinical phase of the disease41,42,. In animal model of PD, MPTP-lesioned nonhuman pri-mates, decreased uptake of 123I-PE2I SPECT was found in asymptomatic animals225.

Because early clinical markers considered separately are neither highly specif-ic, nor sensitive for PD, it has been proposed to develop screening strategies includ-ing combinations of those markers with a neuroimaging marker in order to establish a premotor diagnosis27,226. For example Berendse and co-workers reported a subclinical degeneration of presynaptic dopaminergic system measured with [123I]β-CIT SPECT in hyposmic relatives of PD patients43. Also the recently published study, which was performed by the same group, in a cohort of asymptomatic first-degree relatives of PD patients showed the association between idiopathic olfactory dysfunction and the in-creased risk of developing PD227.

Identification of subjects at risk of developing PD not only allows for the early initia-tion of neuroprotective treatment but also creates the possibility to investigate the neurode-generative process in PD. Taking all data together, DAT imaging may be a sensitive means to detect dopaminergic degeneration in early, and presumably, also in pre-motor phase of PD.

Measuring PD progression

The rate of disease progression in PD has been longitudinally investigated using clini-cal endpoints such as UPDRS-scores89. The interpretation of these data, however, is confounded by symptomatic effects of dopaminergic medications. Although cross-sec-tional necropsy studies estimated the rate of nigral degeneration in PD patients to be 8 to 10-fold that of healthy age matched controls13,72, such a technique could not, by definition, be used to measure progression longitudinally. Imaging, however, may offer the possibility to perform longitudinal assessment. Indeed, several longitudinal DAT imaging studies investigating the rate of PD progression have been performed, the most of them using [123I]β-CIT SPECT. Sequential DAT scans in longitudinal studies (Table 1) showed a mean annual decline in striatal radiotracer uptake ranging from 5 to 13% in PD patients as compare to 0.6-2.5% in healthy controls206,209,210,228-233. These results are in line with the results of longitudinal studies using [18F]dopa PET193,194. Variations in the reported progression rates between studies could be related to different study populations concerning the disease severity, disease duration, duration of follow up, or technical differences (e.g. SPECT versus PET, or analysis method), and whether or not the patients were treated with dopaminergic medication. We examined the change in striatal uptake of 2 different DAT ligands, [123I]β-CIT and [123I]FP-CIT, in sequential SPECT scans in 20 and 50 early stage, de novo PD patients, respectively, and found in both studies a significant 8% reduction of striatal SPECT binding over 1 year229,232. Pirk-er and co-workers206 studied the progression of dopaminergic degeneration in PD over a period of 2 years and found a significant, 7.1% annual decline of [123I]β-CIT SPECT binding in 24 PD patients with short disease duration, while in 12 PD patients with

Chapter 4.2

76

long disease duration the decline in [123I]β-CIT SPECT binding over the same period was not significant. The same trend was shown in a longitudinal [18F]dopa PET study193. The possibility of different rates of progression in subpopulations of PD patients as also suggested by the results of pathological and clinical trials, should be taken into account while designing trials with experimental neuroprotectants.

Although in the cross-sectional studies good correlations were found between the clinical handicap in PD and the extent of reduction in DAT radiotracer uptakes, most of the longitudinal studies failed to show correlations between the rate of progression of dopaminergic degeneration and the changes in clinical parameters over time. The influ-ence of dopaminergic medications on clinical scores might be responsible for that, as well as small study populations, and relatively short follow up periods. Only the CALM-PD imaging study with larger number of subjects included and after follow up of 46 months did show such a correlation205.

None of the above mentioned SPECT studies, however, could show significant subregional differences in the rate of decline of striatal DAT binding. This is in con-

Table 1. Reported rates of annual decline in striatal DAT binding in longitudinal studies.

Refrerence Imaging technique

Mean follow up

Study population

Annual decrease DAT binding

Subregional difference in decline

Staffen et al., 2000 [123I]β-CIT SPECT 15 mnth n=15HY 1 (n=7)HY 2 (n=6)HY 3 (n=2

6.8%6.1%1.3%

no

Nurmi et al., 2000 [18F]CFT PET 2 yr n=8 de novo 13% no

Marek et al., 2001 [123I]β-CIT SPECT 2.3 yr n=32 (HY 1–2.5) 11% no

Winogrodzka et al., 2001 [123I]FP-CIT SPECT 1 yr n=20 de novo 8% no

Chouker et al., 2001 IPT SPECT 2 yr n=8 (HY: 1–3) 5.9% no

Pirker et al., 2002 [123I]β-CIT SPECT 2 yr n=36n=24 earlyn=12 late PD

5%7%2.4%

no

Pirker et al., 2003 [123I]β-CIT SPECT 5 yr n=21 (HY 1–3) 6–7% no

Winogrodzka et al., 2003 [123I]β-CIT SPECT 1 yr n=51 de novo 8% no

Nurmi et al., 2003 [18F]CFT PET 2.2 yr n=12 de novo post. putamen 12.1%ant. putamen 11.9%caudate 8.0%

yes

Abbreviations: HY = Hoehn and Yahr stage

77


trast to the findings of cross-sectional studies showing predominant involvement of contralateral putamen as well as longitudinal [18F]dopa PET data193,194 suggesting that the putaminal decline is faster than that in the caudate. Reviewing PET literature with DAT radiotracers we found one publication investigating subregional differences in the rate of progression of dopaminergic dysfunction in 12 early, de novo PD patients using [18F]CFT PET233 and showing significant differences in annual decline of tracer uptake between the anterior and posterior putamen, suggesting slower progression in the region where disease is already more advanced (posterior putamen). The lower sensitivity of the SPECT method as compared with PET might explain the lack of finding of subregional differences in the decline of radioligand uptake over time mea-sured with DAT SPECT.

Based on the reported rates of disease progression in the longitudinal imaging studies, the estimations of sample sizes required to detect an effect of putative neu-roprotective drugs were provided. We have shown that a 30% protective effect over 2 years could be demonstrated in a study with 238 patients in the treatment and control groups232. Therefore, due to marked between-subject variability in the rate of PD pro-gression, the future studies investigating neuroprotective drugs will need to consider the inclusion of large cohorts with long follow up periods.

Measuring “neuroprotection” in PD

The laboratory data demonstrating the capacity of dopamine agonists to protect dopami-nergic neurons prompted trials investigating their potential disease-modifying effect234,235. On the other hand, concern has been raised about the possible toxic effect of levodopa in PD patients, due to enhancement of oxidative stress as shown in in vitro setting236.

Recently, a prospective double-blind clinical trial has been performed testing the capacity of dopamine agonists to modify the rate of disease progression in de novo PD patients using DAT imaging results as a primary end-point. In 82 early PD patients, ran-domized to receive pramipexole or levodopa, the disease progression was measured with β-CIT SPECT after 22 and 46 months, as a subset of the CALM-PD study205. The initial results showed a non-significant trend in favour of pramipexol. The data presented after 46 months using improved reconstruction analysis technology indicated that the loss of striatal β-CIT uptake from baseline was significantly reduced in the group randomised to pramipexole as compared to the group randomised to levodopa. These results were confirmed by two longitudinal 18F-dopa PET studies: the REAL-PET study and the PEL-MOPET study237,238. In the REAL-PET study, 186 early PD patients were randomised to receive ropinirole or levodopa in a double blind manner, showing significantly 34% less decline in 18F-dopa PET uptake in the ropinirole group after 2 years.

The PELMOPET study examined the disease progression 88 PD patients ran-domised to levodopa and pergolide with 18F-dopa PET and showed a nonsignificant trend in favour of pergolide after 3 years.

Chapter 4.2

7�

The investigators in the above mentioned studies concluded that a smaller decline in radioligand binding implies a (significantly) slower rate of progression of the disease in patients treated with the dopamine agonists as compared with levodopa. Since none of those studies compared the active drug with placebo no distinction could be made be-tween the difference in the rate of loss of radioligand uptake resulting from decrease due to dopamine agonist or increase due to levodopa, or both. The possibility of the use of open-label levodopa in the agonist groups in CALM-PD and REAL-PET studies makes the interpretation of the clinical relevance of these results extra difficult. Interestingly, the patients treated with levodopa did better in terms of clinical end-point (UPDRS). Recently published results of The Parkinson Study Group investigating the effects of levodopa on PD progression showed a significant clinical improvement after 42 weeks in patients receiving levodopa as compared to placebo, but there was a significantly greater decline in β-CIT SPECT binding after 42 weeks in levodopa group as compared to placebo group239. The study medication, though, was not discontinued at the time of the second SPECT scan. Moreover, although the binding ratios were 1.4% lower after placebo as compared to the baseline measurements, this difference was not statistically significant239. Therefore, this study could not prove that DAT scintigraphy per se is able to detect a progress in the decline of dopaminergic neurons in disease. Although it is tempting to draw conclusions about possible neuroprotective actions of dopamine agonists, an important limitation of all these studies so far is the possibility that the study drug itself may directly regulate the radioli-gand binding and affect the validity of using these methods to assess disease progression.

Regulatory effects of drugs and compensatory changes within dopaminergic system

DAT is a protein critically involved in the regulation of the synaptic dopamine levels by removing dopamine from the synapse back into the presynaptic nerve ending. The reup-take of dopamine is regulated via phosphorylation and dephosphorylation processes. Re-cent studies have shown that activation of protein kinase C (PKC) results in a fast decrease of the amount of DAT on the cell membrane (down-regulation or internalization240). Am-phetamines, but also dopamine, are known to activate PKC. Indeed, a recent study showed a down-regulation of DAT after exposure to d-amphetamine or dopamine, which was re-versed by a PKC-inhibitor241. Not only neurotransmitters and drugs, but maybe also hor-mones like estradiol may activate PKC, and consequently influence the amount of DAT on the cell membrane242. Since more and more data are now available on the fast dynamics of DAT expression on the cell membrane, it is not surprising that recent studies performed in humans have shown that age as well as gender, have effect on DAT levels186,243. DAT expres-sion might also be influenced by compensatory changes induced by loss of dopaminergic cells216. In a preclinical stage of PD, in which patients are asymptomatic despite a substan-tial loss of dopamine containing neurons27, one of the possible mechanisms of functional compensation for this neuronal loss may be down-regulation of dopamine reuptake into

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presynaptic nerve terminals216. Animal studies on stimulated dopamine efflux in striatal slices suggest that dopamine reuptake is down-regulated in the denervated striatum244,245. Also in a clinical PET study using 3 different markers of presynaptic dopaminergic function in PD patients, Lee and co-workers suggested that the reduced brain dopamine levels are functionally compensated by down-regulation of DAT215. Therefore, DAT imaging in non-treated PD patients may overestimate nigral cell loss.

All drugs affecting dopaminergic neurotransmission may have the potential to influ-ence DAT regulation (Table 2). The outcomes of DAT imaging studies may, therefore, be influenced by direct competing effects related to drug occupancy of DAT binding site, as well as indirect drug effects changing DAT expression. Most drugs used to treat PD do not have a high affinity for DAT. They might, however, interfere with DAT binding of radioli-gands via modification of it’s expression.

Table 2. Drug effects on DAT binding.

Reference binding Study Ligand Drug Administration Effect

on ligandAllard, et al. Psychopharmacol, 1990

animal(rats)

[3H]GBR-12935 bromocriptineamphetaminecocaine

subchronic nonono

Laruelle, et al.,Synapse, 1993

animal(baboons)

[123I]β-CIT L-dopaamphetamine

acute no↓

Ikawa, et al.Eur J Pharmacol, 1993

animal(rats)

[3H]mazindol L-dopa acutesubchronic

no↑

Gnanalinghan, et al., Brain Res, 1994

animal(rats)

[3H]mazindol L-dopa continousintermittent

↓no

Thibaut, et al.,Neurosc Lett, 1996

animal(mice)

[3H]cocaine[3H]mazondol

L-dopa acute no

Gordon, et al.,Eur J Pharmacol, 1996

animal(rats)

[3H]GBR-12935 L-dopareserpineamantadine

subchronic no↓↑

Moody, et al.,Neuroscie Lett, 1996

animal(rats)

[3H]WIN-35,428 L-dopa subchronic no

Little, et al.,Brain Res, 1996

animal(rats)

[123I]RTI-121[3H]WIN 35428

quinpiroleapomorphine

subchronic nono

Dresel , et al.,Eur J Nucl Med Molec Imaging, 1997

animal(rats)

[99mTc]TRODAT L-dopaapomorphinebromocriptine

acute ↓nono

Rioux, et al.,Mov Disord, 1997

animal(MPTP monkey)

[3H]mazindol L-dopa subchronic ↑

Innis, et al.,Mov Dis, 1999

human [123I]β-CIT L-dopa/ carbidopaL-Selegiline

subchronic nono

Ahlskog, et al.,Mov Dis, 1999

human [123I]β-CIT pergolide subchronic ↑

Nurmi, et al.,J Cereb Blood Flow Metab, 2000

human [18F]CFT L-dopa subchronic no

Guttman, et al.,Neurology, 2001

human [11C]RTI-32 L-dopapramipexol

subchronic ↓↓

Abbreviations : ↑: increase in ligand binding, ↓: decrease in ligand binding

Chapter 4.2

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Acute drug effects

Acute administration of drugs which compete for the DAT binding site, such as co-caine, methylphenidate, amphetamine, bupropion, can displace the radioligand211,246,199,, whereas selective serotonine reuptake inhibitors (SSRI) may increase the radioligand binding to the striatum247. In vitro studies suggest that dopaminergic drugs might influ-ence the expression of DAT216,248,249. In vivo studies in animals show conflicting results. In rats, the acute parenteral administration of dopamine agonists such as apomorphine and bromocriptine did not influence radiotracer binding to DAT199. The acute parenteral administration of high dose levodopa in rats, however, decreased the [99mTC]-TRODAT binding in one study199, but did not influence neither [I123] β-CIT binding in baboons211

nor [3H]cocaine and [3H] mazindol binding in mice246 in two other studies.

(Sub)chronic drug effectsAnimal studies

For trials with PD patients a critical issue is whether chronic or subchronic administra-tion of dopaminergic drugs affects DAT expression.

Gordon and coworkers investigated rats which were treated subchronically with reserpine, amantadine or levodopa and showed that DAT is sensitive to changes in synaptic dopamine contents250. Reserpine treatment which depletes dopamine, caused a significant decrease in [3H]GBR 12935 DAT binding, amantadine which induced do-pamine release caused a significant increase in maximal [3H]GBR binding sites, while treatment with levodopa/carbidopa did not cause any effect on maximal DAT bind-ing. Moody251 investigated the potential role of endogenous dopamine in modulating DAT in rats, chronically intermittently treated with drugs modulating dopamine levels such as levodopa, and found no alterations in radioligand binding to DAT. This was confirmed by Murer and coworkers, who administered levodopa to rats for 6 months and found no effect on DAT expression in either controls or animals with severe 6-hydroxydopamine-induced lesions252. Also in the study of Lavalaye and co-workers on the influence of subchronic (as well as acute) administration of dopaminomimetics (levodopa and pergolide), antipsychotics (olanzepine, risperidone and haloperidol) and antidepressants (fluvoxamine) on [123I]FP-CIT binding in rats no significant effects were found253. In contrast, two other studies reported increased radioligand binding to DAT after chronic levodopa administration in rats254 and in MPTP-lesioned monkeys that have been treated with levodopa for 9 weeks255, whereas another study in rats showed reduced binding after continuous but not intermittent levodopa treatment256.

Chronic treatment with dopamine agonists quinpirole and apomorphine had no effect on DAT binding in rats in one study200, while a non-significant trend toward in-creased striatal DAT binding has been found after 2 to 3 weeks of dopamine agonist administration was found in another study257.

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Taking the evidence from animal studies together it seems that that (sub)chronic exposure to dopaminergic drugs might alter DAT levels. However, the reported direc-tion of the change is not consistent. These inconsistencies might be partly explained by differences in dosing schedule, different durations of exposure to drug and different interval between last dose and DAT measurement. Moreover, the majority of studies have been performed in control animals. As a consequence, these data may be difficult to extrapolate to patients suffering from PD since the influence of medication on DAT expression might be changed in disease.

Human studies

A few studies investigating the effects of dopaminergic treatment on DAT expression have been performed in humans. Innis et al.197 investigated the effects of subchronic treatment with levodopa/carbidopa and selegiline on striatal [123 I]β-CIT SPECT bind-ing in two groups of 8 levodopa-naive PD patients. SPECT scans were performed at baseline, while on medication (after 4-6 weeks) and after withdrawal from medication. No significant modulation in the number of striatal DAT binding was found.

Also a [18F]CFT PET study by Nurmi et al.258 failed to show an influence of le-vodopa on DAT imaging. Ahlskog and co-workers201 reported the effects of dopamine agonist pergolide in 12 advanced, levodopa-treated PD patients on [123I]β-CIT SPECT binding. Scans during 6 weeks pergolide maintenance treatment showed marginally increased [123I]β-CIT uptake (not reaching statistical significance). The authors sug-gested a possibility of minor influence, which might have been statistically significant in a larger patient group.

In a previously published paper232, we attempted to assess the effects of D2 recep-tor agonists (pergolide and pramipexol) on the striatal [123I]β-CIT binding in a group of 9 early PD patients. The patients were imaged twice under the following conditions: 1) while on treatment with D2 receptor agonist and 2) either withdrawn from medication or de novo. The scan-to-scan interval was 2 to 5 weeks. Treatment with D2 receptor ago-nists did not cause significant changes in striatal [123I]β-CIT binding ratios, as measured by serial SPECT images in 2 different conditions.

Guttman and co-workers259 performed a prospective, assessor-blinded, placebo-controlled trial in 30 drug-naive, early PD patients which were randomized to receive 6 weeks of levodopa, pramipexole or placebo. [11C]RTI-32 PET scans were performed at baseline and after 6 weeks (after withholding their study medication overnight). They found a slight but significant 16% reduction in striatal DAT binding after 6 weeks in the levodopa group and to a lesser extent in pramipexole group (7%). The authors con-cluded that the most likely explanation for decreased striatal [11C]RTI-32 PET binding in the levodopa treatment group was that it reflected a drug-induced down-regulation in DAT number or affinity. Indeed, the results of this study raise the possibility of the potential for dopamine-active drugs to regulate DAT. The direction of the change, how-ever, in this study is in contrast with the assumption of up-regulation of DAT in case of

Chapter 4.2

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dopamine excess. One animal study257, however, reports similar results, leaving open the possibility that under some conditions DAT indeed might be down-regulated by treat-ments enhancing dopaminergic function.

The Parkinson Study Group trial investigators recognized the potential confound-ing effects of pharmacologic influences on DAT expression and attempted to investigate this in a small subset of patients within CALM-PD study (n=13). They found no short-term effects of pramipexole (n=7) nor l-dopa (n=6) after 10 weeks of treatment205.

There are several limitations of the human studies that examined the effects of dopaminergic medication. Patients received only subchronic drug treatments (probably to avoid changes due to disease progression) while modulation of DAT binding may re-quire more prolonged exposure to medications. Also, the lack of power might play a role and larger sample sizes may have shown significant drug effects. Innis and coworkers197 assessed the limitations of their study based on sample size using a power calculation and found that the limited sample size in their study may have failed to detect a change of <14% striatal values.

Taken together these results do not show consistent effect of drugs on DAT ex-pression in-vitro or in-vivo, in humans and in animals, although the possibility that DAT neuroimaging studies assess pharmacological drug effects rather than disease pro-gression can not be excluded yet. This is still a point of consideration in future trials of neuroprotective therapy using such techniques.

Reproducibility

For application in longitudinal studies it is critical that the repeated measurements of the radiotracer binding within the same patient are highly reproducible. This issue of reproducibility of DAT imaging was addressed by several SPECT studies. Seibyl and colleagues investigated the reproducibility of [123I]β-CIT SPECT in 7 controls and 7 early PD patients, scanned two times 7–21 days apart. The variability of striatal ratio was 12.8±8.9% in healthy volunteers and 16.8±13.3% for the PD patients40,260 . Later on, Pirker et al.206 reported the mean within-subject variability of striatal β-CIT binding in essential tremor patients to be about 8%. Those results are comparable with the reproducibility of [18F]dopa PET studies in healthy controls193.

Booij et al.196 recognised that operator-dependent ROI technique to assess the striatal binding ratio might introduce some variability in the final result. They assessed the reproducibility of FP-CIT SPECT using a newly developed, fully automatic volume of interest (VOI) protocol establishing a mean test/retest variability of striatal FP-CIT binding in of 7.5% and 7.4% (VOI) in controls and PD patients, respectively, whereas ROI protocol resulted in 7.3% respectively 7.9%. The intraobserver variability using the ROI protocol in this study performed by an experienced investigator was 2.4%.

Nurmi and coworkers studied the reproducibility of [18F]CFT PET 258 in 8 healthy volunteers and 7 PD patients scanned twice approximately 3 months apart.

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In healthy volunteers variability ranged between 1.1% and 4% depending on region studied, whereas in PD variability ranged between 1.9% and 4.1%. The study of Gutt-man and coworkers259 showed quite marked range of interval changes in regional DAT binding with [11C]RTI-32 PET, in the range from +2% to -12% after 6 weeks in non-treated patients.

Present data suggest that the influence of scan-to-scan variability could outweigh the change in imaging index that arises from true disease progression over the short interval of time. Therefore, the value of this method in measuring disease progression in individual patients and/or in small studies might be limited. Reproducibility of imaging depends on several factors, such as choice of the machine, image acquisition, processing and reconstruction, analysis method, and might profit by standardisation of imaging methodology.

Conclusion

In the era of the development of novel neurprotectants two issues are of crucial impor-tance: firstly, the adequate identification of persons at risk/in premotor stage of the dis-ease, and subsequently the ability to evaluate the efficacy of neuroprotectants in clinical trials. The available clinical markers of early/premotor PD are neither highly sensitive, nor specific. DAT imaging with SPECT and PET has been shown to be capable to adequately disclose premotor impairment of the dopaminergic system. For the population-based screening strategies the combinations of clinical markers of early PD such as hyposmia or subtle neurocognitive impairment with neuroimaging markers may be useful.

The clinical measures of PD progression are subjective and do not distinguish between symptomatic and neuroprotective effects of the study drugs. The utility of dopaminergic imaging in measuring the effects of neuroprotection depends on it’s sen-sitivity to disease progression, reproducibility and stability in the environment of com-pounds possibly altering dopaminergic neurotransmission. Extensive evidence from animal and human studies, in healthy controls as well as in PD patients, indicates that DAT imaging is an adequate biomarker for dopamine neuronal terminal integrity and suggest that it may be a suitable tool to monitor PD progression. It also shows an ac-ceptable test-retest reproducibility, though it seems not suitable to measure the disease progression in individual patients because of marked mean test-retest variability of the sequential imaging results.

The possible alteration of DAT binding by effects of drugs remains a concern. Es-pecially preclinical animal studies suggest that dopaminergic drugs might influence the expression of DAT. However, the translation of the results of those studies to human set-ting might be limited not only due to species differences, but also overall short duration of drug treatments, small numbers of tested animals, and varying dose schedules. As of yet, the reviewed literature remains inconsistent about the effects of various manipula-tions of dopaminergic neurotransmission on DAT expression and the reviewed clinical

Chapter 4.2

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studies do not exclude significant effects. Therefore, it remains unclear whether the results of the neuroprotection studies might be due to pharmacologic influence of the study drugs on regulation of proteins involved with dopaminergic neurotransmission, and especially the DAT.

In conclusion, the reviewed evidence supports the use of DAT imaging in early PD detection as well as monitoring PD progression. The crucial issue, however, that needs to be resolved in the future is to determine the extent of potential confounding compen-satory effects of potential neuroprotective agents on the imaging markers.

CHAPTER 5

General discussion and future perspectives

Chapter 5

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Parkinson’s disease (PD) is a common, neurodegenerative disorder responsible for major motor and nonmotor morbidity. It impairs the quality of life of patients and causes substan-tial social and economic burden. The currently available symptomatic treatment, although initially effective, does not satisfactorily control the increasing disability experienced by patients with PD in the long run. Therefore, more effective therapies are required.

A major goal in the development of new treatment strategies for PD is to slow down, or even prevent the progressive degeneration of the nigrostriatal dopaminergic system (and several other CNS neurotransmitter systems) and to reduce functional de-cline in patients. This primary or secondary prevention of the neuronal degeneration in PD could retard the natural course of disease and delay the associated need for medica-tion and the development of serious side effects.

Ingredients, necessary for a successful neuroprotective approach are:1. early disease detection, in order to identify patients at an early stage of pathol-

ogy, when most protective strategies would be of greatest benefit2. insight into the mechanism responsible for neuronal degeneration, in order

to provide therapeutic targets for disease modification3. development of tools that will adequately confirm a neuroprotective effect of the

therapeutic intervention

5.1 Early PD features

From the perspective of disease prevention or delay of disease onset, improvement of early disease detection in PD patients becomes a major goal of clinical research. It is of great interest to identify subjects at risk to develop PD to initiate neuroprotective treatment earlier.

The premotor period (“preclinical window”) during which the neurodegen-erative process in PD occurs while clinical motor dysfunction is still not obvious, would be a good target for neuroprotective therapies. The (bio)markers for early detection of PD may be divided into several categories such as clinical, neuroimag-ing, genetic and biochemical.In the first part of this thesis we focussed on some early clinical features of PD.

In chapter 2.1 we attempted to understand the nature of the tremor in early PD patients by examining the relationship between tremor and rigidity in the same extrem-ity, as well as the relationship between the degeneration of the nigrostriatal pathway and the appearance of tremor. In thirty early, non-medicated PD patients, tremor was as-sessed using accelerometers with spectral analysis of accelerometer recordings. [123I]β-CIT SPECT was used to determine the extent of degeneration of the dopaminergic sys-tem. We hypothesized that the limb rigidity in PD patients influences the expression of tremor, refining the classical distinction between the tremor type and akinetic-rigid type of PD. Several observations were made. Firstly, we found that in de novo PD pa-

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tients, the bulk of tremor and rigidity appear in the same arm, contralaterally to the more affected striatum. Secondly, tremor power accounted for a significant part of vari-ance in the contralateral striatum, suggesting a relation between this PD symptom and the degeneration of the dopaminergic system. Finally, rigidity significantly influenced the expression of tremor in the same arm, however, by correcting for the influence of ri-gidity, the significant contribution of tremor in the variance in the contralateral striatal [123I]β-CIT binding disappeared. Evidently, it is essential for studies focussing on tremor in PD patients to correct for the influence of rigidity.

In chapter 2.2 we focussed on the movement coordination during walking in early PD patients using the dynamical systems approach. The changes of relative co-ordination between arm and leg movements in PD patients were examined during walking on a treadmill with gradually increased, computer-controlled speed. The ex-tent of degeneration of the dopaminergic system was assessed with [123I]β-CIT SPECT. Gradually increasing walking speed results in important transitions in arm and leg movements in the normal human walking mode. We hypothesised that in PD patients, even in the early stage of the disease, the flexibility of adaptation of the coordination patterns during walking will be reduced and accompanied by an increased stability within coordination patterns. Our findings however indicated that the basic coordina-tion patterns are preserved in early PD and that the patients initially are generally able to adapt their arm and leg coordination pattern during walking with externally manip-ulated speed. However, bradykinesia and rigidity as well as the extent of degeneration of the dopaminergic system were associated with a limited adaptive ability (flexibility) in movement coordination.

The above mentioned observations made in a group of early and non-medicated PD patients provided more insight and understanding of the nature of early clinical fea-tures of PD and may enable a more sensitive assessment of the primary and additional features that may even predate the predominant clinical motor syndrome.

Clinical measures such as olfactory testing, mood assessment, neuropsychological tests, autonomous function tests and sleep assessment may be additional clinical param-eters for detecting early PD. Especially olfactory dysfunction might be among the po-tential markers to even identify persons at risk of developing PD. The study of Berendse et al43 indicated subclinical degeneration of the nigrostriatal dopaminergic system, mea-sured with [123I]β-CIT SPECT, in hyposmic, but not normosmic relatives of PD patients. Two year clinical follow up in these subjects showed the development of clinical PD in 10% of PD relatives with hyposmia in combination with strongly reduced [123I]β-CIT SPECT binding at baseline, indicating the association of idiopathic olfactory dysfunc-tion with increased risk of developing PD227. However, as single tests may not be sensitive and specific enough for identifying IPD (for example, olfactory loss also occurs in many other settings than PD, including Alzheimer’s disease), they might need to be used in combination with other tools. The development of a battery with simple, inexpensive but sensitive screening methods seems mandatory. Of course, these tests should measure

Chapter 5

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independent parameters of the disease (for example assessment of olfaction, autono-mous functions, sleep quality, mood and motor function) to identify subjects at risk and should be followed by neuroimaging to determine the extent of neuronal degeneration.

In the meantime, several studies indicated that functional imaging techniques such as DAT SPECT are capable of disclosing early, and even premotor impairment of the dopaminergic system. Patients with hemiparkinsonism showed reduction of the striatal radiotracer uptake not only contralateral to the affected side but also ipsilateral, pointing towards a subclinical damage of the nigral neurons41,42. The combination of transcranial sonography of the substantia nigra and functional neuroimaging of the nigrostriatal system with 18F-Dopa PET recently demonstrated a significant reduction in striatal 18F-Dopa uptake in healthy subjects with substantia nigra hyperechogenicity, suggesting a subclinical reduction in nigral cell count261. Longitudinal follow up studies in larger cohorts will be required with the combinations of these techniques to establish their diagnostic value for the screening of subjects in early or even presymptomatic disease stages.

5.2 Leads for the development of neuroprotec-tive treatment in Parkinson’s disease

In the past few years much has been learned about the etiology and pathogenesis of PD. Research focussed on understanding the mechanisms of cell death in PD has led

to identification of a large variety of pathogenetic factors, including genetic mutations, mitochondrial dysfunction, oxidative stress, inflammatory mediators, excitotoxicity, apoptosis, loss of trophic support and proteasomal dysfunction. The contribution of each of these factors in the neurodegenerative process in PD as well as the primary pathogenic event are not exactly known. In the meantime, therapeutic approaches aimed at correcting these abnormalities are currently being evaluated on their efficacy as disease modifying agents for PD.

In chapter 3 of this thesis we focussed on oxidative stress as one of the possible causative factors of the neuronal degeneration occurring in the substantia nigra in PD. Post-mortem analysis provided information about increased lipid peroxidation, super-oxide dismutase activity and free iron levels as well as reduction in the level of gluta-thione in the substantia nigra of PD patients125. Glutathione, a tripeptide containing glutamate, cysteine and a glycine moiety, is present in all cells of eukaryotic organisms, including humans. Synthesis and degradation of glutathione is catalyzed by γ-glutam-ylcysteine synthetase and γ-glutamyltranspeptidase. Cells use glutathione mainly as an anti-oxidant for the scavenging of organic- and inorganic hydroperoxides and for inac-tivation of endogenous toxic metabolites and xenobiotics through conjugation. These reactions are catalyzed by the enzymes glutathione peroxidase and glutathione trans-ferase, respectively. An upregulation of the activity of γ-glutamyltranspeptidase, which

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is a cell membrane bound protein functioning to increase intracellular glutathione, was reported in the substantia nigra of Parkinson’s disease patients143. This combination of an early decrease in cellular glutathione content, an increase in glutathione ‘uptake’ from the extracellular space in absence of clear alterations in the enzymatic capacity to synthesize or use glutathione for protection against oxidative damage suggests that the degeneration of nigral dopaminergic neurons in PD may be caused by a process in which glutathione is consumed.

In addition to monoamine oxidase-catalyzed enzymatic oxidation, dopamine is also able to undergo autooxidation, a process of oxidation reactions in which highly neurotoxic DA-quinones are produced, with final formation of neuromelanin. The level of these DA-quinones is reported to be increased in substantia nigra in PD. DA-qui-nones are efficiently inactivated through non-enzymatic antioxidants such as ascorbic acid and glutathione and enzymatically by the so-called phase II biotransformation en-zymes: NAD(P)H:quinone oxidoreductase (NQO) and glutathione transferase, both of which are expressed in the human substantia nigra. The activity of these enzymes, can be up-regulated by a large variety of compounds which have been shown to be active both in vitro and in vivo, such as phenolic anti-oxidants, isothiocyanates, nonsteroidal anti-inflammatory drugs (e.g., indomethacin, ibuprofen), and many others.

Thus, given the likely role of dopamine auto-oxidation in the pathogenesis of PD and considering the role of phase II biotransformation enzymes in the detoxication of DA-quinones it is hypothesized that stimulation of pathways involved in the detoxica-tion of DA-quinones in the brain may be an effective means to limit oxidative stress and to provide neuroprotective treatment strategies in PD.

5.3. Dopamine transporter SPECT in monitoring disease progression and measuring “neuropro-tection” in Parkinson’s disease

The search for neuroprotective agents has intensified. Several compounds have already been proposed to have neuroprotective potential based on laboratory research26. As for now, none of them was adequately proven to have an established disease-modifying effect in PD patients. Several approaches have been proposed as measures of the ef-ficacy of neuroprotective interventions. Measuring the time until patients deteriorate as a milestone in disease progression, assessment of clinical features, functional status and the health-related quality of life in PD over time with clinical instruments such as UPDRS, Hoehn and Yahr scale, Schwab and England ADL Scale, PDQ39 were used. The major limitation of using clinical rating scales is the potential confounding symptom-atic effect due to either the study drug itself or dopaminergic agents added over time to maintain patient function. This problem was highlighted by the first “neuroprotection trials”. The trials with Deprenyl and Tocopherol (DATATOP)262 as well as with coen-zyme Q10263 showed the unanticipated symptomatic effects of the study drugs, thereby

Chapter 5

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confounding the interpretations of these studies. This led to a search for a non-clinical, more objective marker of disease progression.

During the past decades SPECT and PET imaging has been used to assess stria-tal changes in dopamine function in patients with PD. Imaging of presynaptic dopa-mine transporter (DAT) with PET and SPECT using various radioligands, such as [123I]β-CIT and [123I]FP-CIT, became a validated tool to assess the integrity of nigros-triatal dopaminergic nerve terminals in vivo. Initial studies with these techniques have shown significant loss of DAT in PD patients as compared to controls36 and good cor-relation with symptom severity37-39. DAT scintigraphy turned out to be sensitive to the loss of dopamine transporters in the very early stages of PD37,38,219 and even before the onset of motor signs27,42,43.

In order to be considered as a measure of neuroprotection, the imaging mark-er has to fulfil several criteria. One of them is it’s sensitivity to the process which is changing with disease progression. In chapter 4.1 we investigated the usefulness of DAT SPECT imaging in longitudinal assessment of disease progression in PD. Two imaging series, 12 month apart were performed with both radioligands in two groups of early stage PD patients: group 1 with 50 PD patients examined with [123I]β-CIT SPECT and group 2 with 20 PD patients examined with [123I]FP-CIT SPECT. We found a significant decrease in radioligand binding ratios between the two consecutive scans at a rate of about 8% per year, in both radioligand groups. These finding is in agreement with the results of recent longitudinal SPECT190,205,206,208,209 as well as [18F]DOPA PET studies 193,195, suggesting the applicability of DAT imaging with both, [123I]β-CIT and [123I]FP-CIT to assess disease progression in (early) PD.

As DAT imaging with SPECT (and PET) can objectively follow loss of dopamine terminal function in PD with time and measure the disease progression, it might pro-vide potential means of monitoring the efficacy of putative neuroprotective agents. An-ticipating on future neuroprotection trials we estimated (based on the reported rates of disease progression in the longitudinal imaging studies; chapter 4.1) the sample sizes required to detect an effect of a putative neuroprotective drug and showed that a 30% protective effect over 2 years could be demonstrated in a study with 238 patients in each group (treatment and control)229,232. Due to the marked between-subject variability in the rate of PD progression, future studies investigating neuroprotective drugs will need the inclusion of large cohorts with long follow up periods.

For application in longitudinal studies and studies assessing neuroprotective properties of therapeutic interventions, it is critical that the repeated measurements of the imaging marker within the same patient are highly reproducible. In chapter 4.2 we critically addressed this issue as well as potential regulatory effects of drugs on DAT imaging results. Reproducibility of imaging depends on several factors, such as choice of the machine, image acquisition, processing and reconstruction, analysis method, and might benefit from standardisation of imaging methodology. Specific DAT binding has traditionally been evaluated using ratios based on region of interest (ROI) techniques.

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The implementation of recent methodological advances may further improve the accu-racy of DAT SPECT. Comparable to statistical parametric mapping used in PET, voxel-by-voxel analyses may be performed with SPECT data. Also obsever independent meth-ods have recently been introduced that allow automatical assessment of displaceable to non-displaceable activity ratios.

The utility of DAT imaging in measuring the effects of neuroprotection depends further on it’s stability in the environments possibly altering dopaminergic neuro-transmission. DAT is a protein critically involved in the regulation of the synaptic dopamine levels by removing dopamine from the synapse back into the presynaptic nerve ending. It´s expression might be influenced by compensatory changes induced by loss of dopaminergic cells216. In a preclinical stage of PD, in which patient’s motor function despite a substantial loss of dopamine containing neurons27 is still intact, it is likely that DAT in remaining neurons is compensatorily down-regulated in an effort to maintain adequate synaptic levels of dopamine216. Due to this possible func-tional compensation for the neuronal loss, DAT imaging in non-treated PD patients may overestimate nigral cell loss.

Whether DAT imaging might be influenced by pharmacological effects of drugs is still a matter of debate. Several animal and a few human studies investigating the ef-fects of medications on DAT measurement provided conflicting results raising the pos-sibility of the potential for dopamine-active drugs to regulate DAT expression. Short term treatment with D2 receptor agonists in a subgroup of PD patients in our study did not cause significant alterations in the expression of striatal dopamine transporters as measured using [123I]β-CIT SPECT232. The future carefully controlled studies with repeated imaging after dose titration and after withdrawal of different types of medica-tions at the end of trial have to provide the consensus on the possible effect and the direction of the change in striatal DAT levels caused by drugs.

In the meantime, several trials have already attempted to identify possible neuro-protective effects of drugs using the change in neuroimaging biomarker over time237-239. The well-known hypothesis, that dopamine receptor agonists may extert a disease-mod-ifying, protective effect in PD, as opposed to levodopa which might be neurotoxic to remaining dopaminergic neurons234-236, was tested in the REAL-PET study237 (with rop-inirole versus levodopa, using change in 18F-dopa PET uptake as primary endpoint) and the CALM-PD study239 (with pramipexole versus levodopa, usingng change in striatal [123I]β-CIT SPECT uptake as primary endpoint). Both studies demonstrated a reduced rate of decline of nigrostriatal function in patients receiving dopamine agonists com-pared with levodopa, which might imply a protective effect of agonist or a toxic effect of levodopa. However, it has been suggested that both imaging parameters, DAT SPECT as well as 18F-dopa PET, could have been influenced by direct pharmacologic effects of treatments and the debate raised about the validity of those methodologies as a method of assessment of the neuroprotective properties of drugs which are being tested220-222. More research is required to elucidate these issues.

Chapter 5

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Taken together, although there are good arguments for seeking alternatives to clin-ical assessment because of variability of the clinical measurement, DAT imaging might not yet provide an absolutely ideal measure of terminal density being rather a marker of nerve terminal function potentially influenced by compensatory mechanisms. Despite this, it should provide a valuable adjunct to clinical data in the assessment of disease modifying therapies in PD.

Concluding remarks

The development of therapeutic interventions capable of more effective control of the disease and influencing its course is a major priority of the current research in PD. Based on various etiopathogenetic factors identified to date, several agents with pos-sible disease modifying potential have been suggested for clinical testing. Which pa-tients/individuals should be included in such trials and how to measure the disease modifying effects of the drugs tested, remain to be determined. The concept of pre-clinical/premotor phase in PD offers an attractive target for such interventions with primary disease prevention as an ultimate goal.

Functional neuroimaging of the dopamine transporters with SPECT (and PET) provides a marker of the integrity of the presynaptic dopaminergic system in PD. It correlates with severity of motor dysfunction and shows progressive decline over time. Additionally, it might also be considered to be part of a preclinical screening strategy to identify the individuals at risk of developing PD. Whether this technique is a suitable marker to detect putative neuroprotective effects in PD however, is ques-tionable. At present, it seems that neither the clinical nor the imaging markers used separately are sufficient to accurately measure the effect of intervention on the rate of PD progression. In future studies it might be useful to evaluate multiple primary endpoints reflecting the combined assessment of different dimensions of disease pro-gression in PD.

CHAPTER 6

Summary

Chapter 6

�4

Parkinson´s disease is a severe and progressive neurodegenerative disorder pathologi-cally characterized by preferential loss of neurons in the substantia nigra. The etiological mechanism responsible for this neuronal degeneration remains largely unknown. The cardinal clinical features are rigidity, bradykinesia, resting tremor and postural instabil-ity. The most of patients eventually face significant functional deterioration that cannot adequately be controlled with available medications. Better long term management of PD includes development of therapies which would be able to prevent or slow down the progressive neuronal degeneration and clinical disability. In seeking for such a therapy several issues are of crucial importance. Besides the need for understanding of the un-derlying pathological mechanism responsible for cell death, in order to be able to de-velop promising neuroprotective agents, it is imperative to be able to detect patients at early stages of the disease and to identify the subjects at risk. Only administered as early as possible in the disease course the neuroprotective therapies can be effective. In addition, it is necessary to define reliable endpoints for clinical trials that reflect disease progression, in order to determine whether the therapeutic intervention indeed influ-enced the disease course.

The aim of this thesis was first to address the early clinical (motor) features of PD. Using the tools from dynamical theory of movement coordination we tried to obtain more insight in the nature of the clinical signs of PD early in its course, which might be valu-able in the early detection of the disease. Next, we highlighted the etiopathogenetic mechanisms of PD within the frame of oxidative stress theory and discussed leads for the development of putative neuroprotective strategies. In search for a tool which could be used to measure the effects of putative neuroprotective interventions we investigated the utility of [123I]β-CIT and [123I]FP-CIT SPECT in the assessment of the rate of pro-gression of dopaminergic degeneration in early PD patients.

In chapter 1 general background information on Parkinson’s disease (pathology, patho-genesis, clinical features, diagnosis, natural course, prognosis and therapy) is provided. The need for development of therapies which could prevent further dopaminergic de-generation and thereby slow down or halt the disease progression is underlined. The possibilities to identify patients in early or even preclinical disease stage are discussed.

In chapter 2 the clinical features of early PD such as tremor, rigidity and bradykinesia, are analyzed, partly from the perspective of dynamical systems theory. In chapter 2.1 we attempted to disclose the nature of the resting tremor in PD and it’s relationship with the disruption of the dopaminergic pathway, assessed with [123I]β-CIT SPECT, in early, de novo PD patients. We found that rigidity significantly influenced the expression of tremor in the same arm. However, when confounding influence of rigidity was taken into account, no significant correlation was found between striatal radioligand bind-ing and the degree of tremor. This suggests that other pathophysiological mechanisms

�5

Summary

might be involved in the development of resting tremor in PD. The appearance of domi-nant tremor and rigidity in the same extremity makes is important to correct for the influence of rigidity in settings where the resting tremor in PD patients is investigated.

In chapter 2.2 the coordination patterns in early, de novo PD patients during walking were investigated using dynamical systems approach. The walking speed was systemati-cally manipulated. We hypothesized that classical PD symptoms such as bradykinesia and rigidity reduce the flexibility of coordination patterns of arm and leg movements during walking with systematically manipulated speed even in the early stage of the disease. Our findings indicated that early-stage PD patients were generally, to a certain degree, able to adapt their arm and leg coordination during walking with externally manipulated speed. However, this adaptation was markedly limited by rigidity as well as bradykinesia and it’s extent was related to the degeneration of the presynaptic dopami-nergic pathway as expressed by [123I]β-CIT SPECT.

In chapter 3 etiopathogenic mechanisms of PD are discussed along the line of the oxida-tive stress hypothesis and within this frame the possible targets for neuroprotective treat-ment in PD are proposed. Especially the role of glutathione (loss) in PD, phase II bio-transformation enzymes, dopamine auto-oxydation and detoxication are highlighted.

Dopamine transporter (DAT) imaging with PET and SPECT are valuable tools for the assessment of the integrity of presynaptic dopaminergic nerve terminals in PD. In search for a reliable method to detect the rate of disease progression in PD, that can be used to measure the effects of putative protective therapies we focused on DAT SPECT.

In chapter 4.1 we describe the results of longitudinal assessment of disease pro-gression in early stage, de novo PD patients with SPECT and two DAT radioligands, [123I]β-CIT and [123I]FP-CIT. SPECT imaging with both radioligands shows comparable imaging quality, however the faster kinetics of [123I]FP-CIT allows for image acquisi-tion as early as 3 h post-injection (versus 24 h post-injection in case of [123I]β-CIT). Our results indicated the mean annual rate of dopaminergic degeneration in early stage PD to be about 8%, which is much faster than in normal aging. We concluded that SPECT imaging with both radioligands, [123I]β-CIT and [123I]FP-CIT, is an adequate tool to measure the rate of disease progression in PD.

Then we investigated the influence of dopaminergic medication (dopamine ago-nists) on SPECT DAT binding measures. In a subgroup of PD patients sequentially im-aged while on treatment with D2 receptor agonists and drug-naïve or withdrawn from D2 agonist treatment no significant changes in the binding of [123I]β-CIT to striatal dopamine transporters was found, suggesting the stability of DAT imaging upon this dopaminergic medication. Additionally, we performed sample size calculations for future studies on the evaluation of neuroprotective treatments.

Chapter 6

�6

In chapter 4.2 we reviewed the evidence on the utility of DAT imaging in early PD de-tection, as a biological marker of PD progression as well as a tool to monitor the effects of putative neuroprotective drugs. Since it is clear that, in neuroprotective trial design, the biomarker should reflect a biological process that changes with progression of PD and that it’s measurement should be reproducible and not affected by the study drug or by symptomatic treatments for PD, we critically addressed the issues of sensitivity, reproducibility as well as potential regulatory effects of drugs on DAT imaging results.

Chapter 5 contains concluding remarks and discusses the directions for future research.

CHAPTER 7

Samenvatting

Chapter 7

��

De ziekte van Parkinson is een chronische, degeneratieve aandoening, gekenmerkt door een progressief verlies van, onder andere, dopamine producerende neuronen in de substantia nigra. De oorzaak van het afsterven van deze hersencellen is nog altijd onbekend. De klassieke verschijnselen van de ziekte van Parkinson bestaan uit rigidi-teit, bradykinesie, rusttremor en loop-en evenwichtsstoornissen. Na verloop van tijd leiden deze verschijnselen bij de meeste patiënten tot toenemende functionele invali-diteit. De huidige therapeutische opties, die op dit moment uitsluitend symptomatisch zijn, bieden hiervoor onvoldoende oplossing. Een optimale lange termijn behandeling van de ziekte van Parkinson is dan ook gericht op het ontwikkelen van therapieën waarmee het progressieve degeneratieve proces en daarmee een toenemende klinische handicap vertraagd of zelfs voorkomen zouden kunnen worden (neuroprotectie). Het verkrijgen van inzicht in de onderliggende pathogenese van het ziekteproceses is daarbij essentieel. Omdat de neuroprotectieve therapie alleen effectief kan zijn wan-neer het zo vroeg mogelijk wordt toegepast in het ziekteproces, is een vroege (bij voorkeur presymptomatische) detectie van de ziekte van Parkinson van groot belang. Daarnaast is het van belang betrouwbare eindpunten te definiëren waarmee in klini-sche trials de effecten van therapeutische interventies op de progressie van de ziekte gemeten kunnen worden.

Het eerste deel van dit proefschrift concentreert zich op de waarde van klassieke klini-sche (motorische) verschijnselen bij de vroegdiagnostiek van de ziekte van Parkinson. De invloed van tremor, rigiditeit en bradykinesie op coördinatie processen tijdens het lopen wordt bestudeerd vanuit het perspectief van de theorie over dynamische syste-men. Vervolgens worden de etiopathogenetische mechanismen die mogelijk ten grond-slag liggen aan het ontstaan van de ziekte van Parkinson besproken. In de context van de oxidatieve stress theorie worden de aanknopingspunten en voorwaarden voor de ontwikkeling van neuroprotectieve strategieën beschreven. Tenslotte, op zoek naar een meetinstrument voor de objectieve beoordeling van de effecten van geneesmiddelen op de progressie van de ziekte, wordt de toepassing van dopamine transporter SPECT techniek onderzocht bij het vervolgen van het degeneratieve proces in tijd, bij patiënten in de vroege fase van de ziekte van Parkinson.

Hoofdstuk 1 beschrijft algemene achtergronden van de ziekte van Parkinson. Het be-lang van de ontwikkeling van therapieën die de degeneratie van dopaminerge neuronen en daarmee progressie van de ziekte zullen vertragen, evenals de ontwikkeling van ef-fectieve vroegdiagnostiek worden benadrukt.

In hoofdstuk 2 worden de motorische verschijnselen zoals tremor, rigiditeit en brady-kinesie bij patiënten in vroege fase van de ziekte van Parkinson geanalyseerd vanuit het perspectief van dynamische systemen theorie. In hoofdstuk 2.1 wordt ingegaan op de relatie tussen de rusttremor en de mate van de degeneratie van het dopaminerge systeem

��

Samenvatting

bij vroege Parkinson patiënten, gemeten met [123I]β-CIT SPECT enerzijds, en de rela-tie tussen de rusttremor en andere verschijnselen zoals rigiditeit, anderzijds. Rigiditeit bleek de expressie van de rusttremor in dezelfde extremiteit significant te beïnvloeden. De relatie tussen de mate van de rusttremor en de striatale [123I]β-CIT SPECT binding blijkt echter niet significant, ook na correctie voor deze invloed van de rigiditeit op de expressie van de tremor, hetgeen suggereert dat andere pathofysiologische mechanis-men betrokken zijn bij het ontstaan van de Parkinson tremor. De resultaten tonen aan dat rekening houden met de invloed van rigiditeit van belang kan zijn bij het bestuderen van de tremor bij de ziekte van Parkinson.

In hoofdstuk 2.2 worden de coördinatiepatronen van patiënten met vroege ziekte van Parkinson bestudeerd tijdens het lopen, waarbij de loopsnelheid systematisch wordt gemanipuleerd. De resultaten suggereren dat patiënten tot op zekere hoogte in staat zijn om tijdens het lopen met extern gemanipuleerde snelheid de coördinatie tussen arm- en beenbewegingen te adapteren. Deze adaptatie wordt echter beperkt door de klassieke verschijnselen van de ziekte van Parkinson, zoals bradykinesie en rigiditeit, en correleert met de mate van de degeneratie van het dopaminerge systeem, zoals gemeten met [123I]β-CIT SPECT.

De exacte pathogenese van de ziekte van Parkinson is bij de meeste patiënten onbekend. Oxidatieve stress werd verondersteld één van de mogelijke oorzakelijke factoren te zijn in de degeneratie van de substantia nigra. Hoofdstuk 3 bespreekt de mogelijke targets voor neuroprotectieve behandeling van de ziekte van Parkinson in de context van de oxidatieve stress hypothese.

Functionele neuroimaging van de dopamine transporter met PET en SPECT is een techniek die gebruikt wordt bij de beoordeling van de integriteit van het dopaminerge systeem bij de ziekte van Parkinson. Op zoek naar een betrouwbare methode voor de beoordeling van de progressie van de ziekte van Parkinson, evenals voor de beoordeling van de effectiviteit van een (toekomstige) neuroprotectieve therapie werd DAT SPECT onderzocht.

In hoofdstuk 4.1 worden longitudinale studies beschreven waarin de progressie van de ziekte van Parkinson bij de novo patiënten wordt gemeten middels SPECT tech-niek met twee radioliganden voor de dopamine transporter, [123I]β-CIT and [123I]FP-CIT. SPECT scanning met beide radioliganden toont een vergelijkbare imaging kwa-liteit, echter de snellere kinetiek van [123I]FP-CIT maakt acquisitie na 3 uur mogelijk (versus 24 uur na injectie in het geval van [123I]β-CIT). De resultaten laten verder zien dat de jaarlijkse progressie van de dopaminerge degeneratie in de vroege fase van de ziekte van Parkinson ongeveer 8% bedraagt. Dit is aanzienlijk sneller in vergelijking met normale veroudering. Hiermee is aangetoond dat SPECT imaging met beide radi-oliganden, [123I]β-CIT and [123I]FP-CIT, een goede methode is voor de beoordeling van de progressie van de ziekte van Parkinson.

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Ter beoordeling van de stabiliteit van de resultaten van DAT SPECT imaging onder invloed van dopaminerge medicatie werd het (sub)acute effect van dopamine agonisten (pegolide en pramipexol) op de SPECT radioligand binding bestudeerd. In een groep patiënten, sequentieel gescanned tijdens gebruik van een D2 receptor agonist en medi-catie-vrij, werden geen significante verschillen gevonden tussen deze twee condities in de striatale [123I]β-CIT binding aan de dopamine transporters.

Op basis van de in de longitudinale studies gerapporteerde snelheid van de pro-gressie van de dopaminerge degeneratie werd berekend hoe groot de behandel- en con-trole groepen dienen te zijn voor een “neuroprotectie trial”. Om voor een hypothetisch middel in 2 jaar een protectief effect van 30% aan te tonen zullen er 238 patiënten gein-cludeerd moeten worden in beide groepen.

In hoofdstuk 4.2 wordt een overzicht gegeven van het huidige bewijs betreffende de bruikbaarheid van DAT imaging in vroegdiagnostiek, als een marker voor ziekte pro-gressie en als een methode voor de beoordeling van de effectiviteit van toekomstige neuroprotectieve therapieën. Speciale nadruk is gelegd op de sensitiviteit, reproduceer-baarheid en mogelijke effecten van geneesmiddelen op DAT imaging.

Tenslotte, worden in hoofdstukken 5 en 6 enkele afsluitende opmerkingen en suggesties voor toekomstig onderzoek geplaatst en worden de resultaten van de beschreven studies kort samengevat. Effectieve behandeling van de ziekte van Parkinson houdt in dat er (vroeg) ingegrepen kan worden in het beloop van de ziekte. Zowel klinische als imaging markers kunnen gebruikt worden voor de preklinische/premotorische detectie van de ziekte en voor de beoordeling van de effectiviteit van therapeutische interventies. Aan-vullend onderzoek is nodig om meer inzicht te krijgen welke markers (of combinatie van markers) het beste ingezet kunnen worden voor deze doelen.

CHAPTER 8

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226. Stiasny-Kolster K, Doerr Y, Moller JC, Hoffken H, Behr TM, Oertel WH, Mayer G. Combination of “idiopathic “ REM sleep behaviour disorder and olfactory dysfunction as possible indicator for alpha-synucleinopathy demonstrated by dopamine transporter FP-CIT-SPECT. Brain 2005; 128:126–137

227. Ponsen MM, Stoffers D, Booij J, van Eck-Smit BLF, Wolters EC, Berendse HW. Idiopathic hyposmia as a preclinical sign of Parkinson’s disease. Ann Neurol 2004; 56:173–181

228. Marek K, Innis R, van Dyck C, Fussell B, Early M, Eberly S, Oakes D, Seibyl J. [123I]beta-CIT SPECT imaging assessment of the rate of Parkinson’s disease progression. Neurology 2001; 57: 2089–2094

229. Winogrodzka A, Bergmans P, Booij J, van Royen EA, Janssen AG, Wolters EC. [123I]FP-CIT SPECT is a useful method to monitor the rate of dopaminergic degeneration in early-stage Parkinson’s dis-ease. J Neural Transm. 2001; 108:1011–1019

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231. Pirker W, Holler I, Gerschlager W, Asenbaum S, Zettinig G, Brucke T. Measuring the rate of progres-sion of Parkinson’s disease over a 5-year period with β-CIT SPECT. Mov Dis 2003; 18:1266–1272

232. Winogrodzka A, Bergmans P, Booij J, van Royen EA, Stoof JC, Wolters EC. [123I]beta-CIT SPECT is a useful method for monitoring dopaminergic degeneration in early stage Parkinson’s disease. J Neurol Neurosurg Psychiatry. 2003; 74:294–298

233. Nurmi E, Bergman J, Eskola O, Solin O, Vahlberg T, Soninen P, Rinne J. Progression of dopaminer-gic hypofunction in striatal subregions in Parkinson’s disease using [18F]CFT PET. Synapse 2003; 48:109–115

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253. Lavalaye J, Knol RJ, de Bruin K, Reneman L, Janssen AG, Booij J. [123I]FP-CIT binding in rat brain after acute and sub-chronic administration of dopaminergic medication Eur J Nucl Med 2000; 27:346–349

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Publications

Winogrodzka A, Booij J, Wolters EC. Disease-related and drug-induced changes in dopamine transporter expression might undermine the reliability of imaging stud-ies of disease progression in Parkinson’s disease. Parkinsonism & Related Disorders 2005;11(8):475–84

Winogrodzka A, Wagenaar RC, Booij J, Wolters EC. Rigidity and bradykinesia reduce interlimb coordination in Parkinsonian gait. Arch Phys Med Rehabil. 2005;86(2):183–9

Winogrodzka A, Bergmans P, Booij J, van Royen EA, Stoof JC, Wolters EC. [(123)I]beta-CIT SPECT is a useful method for monitoring dopaminergic degeneration in early stage Parkinson’s disease. J Neurol Neurosurg Psychiatry 2003;74(3):294–8

Winogrodzka A, Booij J and Wolters E Ch. J Neurol Neurosurg Psychiatry. 2003;74(10):1447

Winogrodzka A, Bergmans P, Booij J, van Royen EA, Janssen AG, Wolters EC. [123I]FP-CIT SPECT is a useful method to monitor the rate of dopaminergic degeneration in early-stage Parkinson’s disease. J Neural Transm. 2001;108(8–9):1011–9

Winogrodzka A, Wagenaar RC, Bergmans P, Vellinga A, Booij J, van Royen EA, van Emmerik RE, Stoof JC, Wolters EC. Rigidity decreases resting tremor intensity in Par-kinson’s disease: A [(123)I]beta-CIT SPECT study in early, nonmedicated patients. Mov Disord. 2001;16(6):1033–40

Vlaar AMM, Nijs de T, Kessels AGH, Vreeling FW, Winogrodzka A, Mess WH, Tromp SC, Kroonenburgh van MJPG, Weber WEJ. Diagnostic value of FP-CIT and IBZM SPECT scans in 248 patients with parkinsonian syndromes. Submitted

Publications

Chapter 8

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Vlaar AMM, Bouwmans AEP, Kroonenburgh van MJPG, Mess WH, Tromp SG, Wuis-man PGWM, Kessels AGH, Winogrodzka A, Weber WEJ. Protocol of a prospective study on the diagnostic value of Transcranial Duplex Scanning of the substantia nigra in patients with parkinsonian symptoms. BMC Neurology, accepted

Stoffers D, Booij J, Bosscher L, Winogrodzka A, Wolters EC, Berendse HW. Early-stage [123I]beta-CIT SPECT and long-term clinical follow-up in patients with an initial diag-nosis of Parkinson’s disease. Eur J Nucl Med Mol Imaging. 2005;32(6):689–95

Booij J, Bergmans P, Winogrodzka A, Speelman JD, Wolters EC. Imaging of dopamine transporters with [123I]FP-CIT SPECT does not suggest a significant effect of age on the symptomatic threshold of disease in Parkinson’s disease. Synapse. 2001;39(2):101–8

Wolters EC, Francot C, Bergmans P, Winogrodzka A, Booij J, Berendse HW, Stoof JC. Preclinical (premotor) Parkinson’s disease. J Neurol. 2000;247 Suppl 2:II103–9

Stoof JC, Winogrodzka A, van Muiswinkel FL, Wolters EC, Voorn P, Groenewegen HJ, Booij J, Drukarch B. Leads for the development of neuroprotective treatment in Parkinson’s disease and brain imaging methods for estimating treatment efficacy. Eur J Pharmacol. 1999;375:75–86

Booij J, Tissingh G, Winogrodzka A, van Royen EA. Imaging of the dopaminergic neu-rotransmission system using single-photon emission tomography and positron emis-sion tomography in patients with parkinsonism. Eur J Nucl Med. 1999;26(2):171–82

Van Emmerik RE, Wagenaar RC, Winogrodzka A, Wolters EC. Identification of axial rigid-ity during locomotion in Parkinson disease. Arch Phys Med Rehabil. 1999;80(2):186–91

Booij J, Tissingh G, Winogrodzka A, Stoof JC, van Royen EA. Degeneratie van dopaminerge neuronen bij M. Parkinson. Nerderlands Tijdschrift voor Neurologie 1999;4:220–225

Tissingh G, Bergmans P, Booij J, Winogrodzka A, van Royen EA, Stoof JC, Wolters EC. Drug-naive patients with Parkinson’s disease in Hoehn and Yahr stages I and II show a bilateral decrease in striatal dopamine transporters as revealed by [123I]beta-CIT SPECT. J Neurol. 1998;245(1):14–20

Tissingh G, Booij J, Bergmans P, Winogrodzka A, Janssen AG, van Royen EA, Stoof JC, Wolters EC. Iodine-123-N-omega-fluoropropyl-2beta-carbomethoxy-3beta-(4-iod ophenyl) tropane SPECT in healthy controls and early-stage, drug-naive Parkinson’s disease. J Nucl Med. 1998;39(7):1143–8

11�

Booij J, Habraken JB, Bergmans P, Tissingh G, Winogrodzka A, Wolters EC, Janssen AG, Stoof JC, van Royen EA. Imaging of dopamine transporters with iodine-123-FP-CIT SPECT in healthy controls and patients with Parkinson’s disease. J Nucl Med. 1998;39(11):1879–84

Booij J, Tissingh G, Winogrodzka A, Boer GJ, Stoof JC, Wolters EC, van Royen EA. Practical benefit of [123I]FP-CIT SPET in the demonstration of the dopaminergic defi-cit in Parkinson’s disease. Eur J Nucl Med. 1997;24(1):68–71

Tissingh G, Booij J, Winogrodzka A, van Royen EA, Wolters EC. IBZM- and CIT-SPECT of the dopaminergic system in parkinsonism. J Neural Transm Suppl. 1997;50:31–7

Tissingh G, Bergmans P, Winogrodzka A, Stoof JC, Wolters EC, Booij J, Van Royen EA. Nigrostriatal dopaminergic imaging with iodine-123-beta CIT-FP/SPECT and fluorine-18-FDOPA/PET. J Nucl Med. 1997;38(8):1271–2

Tissingh G, Bergmans P, Booij J, Winogrodzka A, Stoof JC, Wolters EC, Van Royen EA. [123I]beta-CIT single-photon emission tomography in Parkinson’s disease reveals a smaller decline in dopamine transporters with age than in controls. Eur J Nucl Med. 1997;24(9):1171–4

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Dankwoord

Iedereen die in welke vorm dan ook heeft bijgedragen aan het tot stand komen van dit werk wil ik hierbij hartelijk bedanken.

Mijn speciale dank gaat uit naar de patiënten met de ziekte van Parkinson die met grote inzet, belangstelling en vertrouwen hebben meegedaan aan verschillende onder-zoeken beschreven in dit proefschrift.

Dankwoord

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parkinson’s disease monitoring early diagnosis and disease ... dissertation.pdf · op donderdag 8 november 2007 om 13.45 uur in het auditorium van de universiteit,