Positron-emission tomography molecular imaging of glia and myelin in drug discovery for multiple sclerosis.

Review

Positron-emission tomography molecular imaging of glia and myelin in drug discovery for multiple sclerosis

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Introduction

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Molecular imaging with PET

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Imaging microglia

Paul M Matthews† & Gourab Datta

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Molecular imaging of astrocyte

†

Imperial College London, Division of Brain Sciences, Department of Medicine, London, UK

activation

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Kainan University on 04/14/15 For personal use only.

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Molecular imaging of brain myelin

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Conclusion

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Expert opinion

Introduction: Therapies acting on glial cells are being explored for new drug development for multiple sclerosis. Molecular imaging using positronemission tomography (PET) could address relevant questions in early phase clinical trials. Areas covered: In this article, the authors critically review human PET methods that can be applied in specialised centres for imaging activated microglia and astrocytes and myelin. Expert opinion: Strengths of PET lie in the molecular selectivity, sensitivity and potential for absolute quantitation. Even now, translocator protein PET radioligands could be used in exploratory studies for interventions targeting brain microglial activation. The clinical and neuropathological meaningfulness of signal from PET radioligands reporting on astrocyte activation through cellular expression of either monoamine oxidase B or the I2-imidazoline receptor or metabolism of [11C]acetate can now explored. [11C] N-methyl-4,4¢-diaminostilbene, a PET marker for myelin, could soon enter first human trials. However, use of any of these PET glial markers demands a well-focused hypothesis and a commitment to validation in the context of use. Enhanced access to these radioligands, standardisation of analyses and lowering the costs of using them are needed if their full promise is to be realised. Keywords: astrocytes, drug development, imaging, microglia, myelin, positron-emission tomography, radioligand Expert Opin. Drug Discov. [Early Online]

1.

Introduction

Drug development for multiple sclerosis (MS) is increasingly focusing on opportunities for therapeutic modulation of glial cells. There is an increasing interest in developling therapies that are capable of limiting proinflammatory activity of microglia [1]. Enhancement of oligodendroglial regeneration and remyelination are being explored as neuroprotective strategies [2]. Control of astroglial responses to injury also may be fundamental to both myelin and neurite regeneration after neuroinflammation [3] in MS and related disorders. There are several well-accepted MRI measures of disease activity that are suitable for some applications [4]. However, there is an unmet need for additional, direct pharmacodynamic measures of glial activation or myelin. Our rationale for this is briefly set out in the remainder of this introduction, where we will review magnetic resonance technologies to clarify the unmet need. We then will discuss the emerging positron-emission tomography (PET) molecular imaging methods that promise more direct, quantitative measures for glial and myelin evaluation. This expert opinion will outline the progress towards achieving this promise and will suggest areas

10.1517/17460441.2015.1032240 © 2015 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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P. M. Matthews & G. Datta

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Molecularly selective positron-emission tomography (PET) imaging markers of activated microglia and astrocytes and of myelin could support some pharmacodynamic proofs of principle in early phase drug development. PET radioligands targeting the 18 kDa mitochondrial translocator protein (TSPO) are sensitive to microglial (and astroglial) activation with disease or treatment effects. Brain [11C]acetate uptake assesses astroglial metabolism and is sensitive to disease-associated changes with multiple sclerosis. Activated astrocytes can be imaged clinically through their expression of monoamine oxidase-B with [11C] deuterium-L-deprenyl. Strong preclinical proofs of principle for molecularly selective markers of activated astrocytes ([11C]BU99008) and myelin ([11C]MeDAS) promise additional radioligands for future clinical pharmacodynamic studies.

This box summarises key points contained in the article.

that need research and development if PET is to become a useful and practical complement to MRI in drug development for MS. Imaging glial activation with magnetic resonance spectroscopy

1.1

Magnetic resonance spectroscopy (MRS) relies on the same instrumentation as MRI, but detects signals at lower abundance than water. It differentiates molecules (typically brain metabolites present in concentrations that are orders of magnitude lower than brain water) on the basis of differences in the relative frequencies of magnetic resonance of their constituent nuclei. Because the signals are orders of magnitude lower than for MRI, the spatial resolution of MRS is lower than that of MRI. Multiple small molecules can be detected with a single observation based on their intrinsic (e.g., with 1H MRS) or selectively chemically enriched (e.g., with 13C MRS) signal. Brain myo-inositol concentration assessed with 1H MRS is a marker of glial cell activation and proliferation [5]. Concentrations of myo-inositol are greater in astrocytes than in neurons. Consequently, myo-inositol concentration is increased in brain white matter of people with MS [6] and in acute MS lesions relative to white matter that appears normal by conventional MRI [7]. Consistent with interpretation of elevated myo-inositol concentration as a marker of microglial activation in people with MS, higher brain concentrations are associated with short-term progression of disability [8,9]. Nonetheless, evidence supporting the sensitivity and specificity of MRS for microglial activation is still limited. Clinical spectroscopic imaging has only modest spatial resolution, confounding its quantitative interpretation in a multifocal disease such as MS. An alternative approach relies on the relatively high expression of the monocarboxylate transporter (MCT) in astrocytes. 2

This allows them to metabolise acetate preferentially, the rate of which provides an index of astrocyte density and activation. Metabolism can be measured using 13C MRS with administration of acetate enriched at either the C1 or C2 position. Rates of incorporation of the 13C label into brain metabolites after intravenous infusion of enriched acetate provides an index of glial cell abundance. A pilot application in patients with Alzheimer’s disease has demonstrated an elevated metabolic rate, consistent with glial activation [10]. Applications to MS have not been demonstrated yet, to our knowledge. The method shares with the analogous PET approach (see Section 4.1) a need to frame any pharmacodynamic hypothesis in terms of astrocyte metabolic rate. The 13C signal is low on clinical 3T MRI scanners even with isotope enrichment, limiting both precision of the measurement and spatial resolution. In general, MRS is better suited for evaluation of a generalised inflammatory process because of its limited spatial resolution, although novel acquisition and processing methods have substantially advanced the potential for MRS imaging [11]. Applications usage of ultra-high-field 7T magnets also promises an improvement in the spatial resolution, as MRI signal to noise improves with higher field strengths. The methods are quantitative and can provide absolute measures of concentration in ideal circumstances, but there are many challenges in obtaining accurate and precise data, for example, because of partial volume effects arising from the low spatial resolution and the need to account for magnetic resonance relaxation effects. Non-conventional MRI for brain myelin imaging Although not routinely used in clinical diagnostics or monitoring, proton MRI measures based on water relaxation rates [12,13], magnetisation transfer [14] or diffusion tensor imaging [15] are sensitive to brain or spinal cord myelin. However, they also are indirect and are measures of ways in which changes in myelin content influence the water magnetic resonance signal, rather than direct measures of myelin. Currently, their interpretation relies on empirical relationships established by using animal models and studies of tissue ex vivo. Although each of these methods can be highly precise, they provide measures only of relative differences in myelin content. It is not possible to relate measures acquired using the three methods to each other in any generally applicable, quantitative fashion, although all show changes with demyelination. This reflects fundamental differences in the principles on which the methods are based. Nonetheless, they have the advantage of providing images with the spatial resolution of conventional clinical brain imaging (i.e., substantially greater resolution than either PET or MRS) and can be acquired on usual clinical high-field MRI systems. MRI of myelin based on water relaxation rate is made possible because water associates reversibility with the exposed polar amino acids of myelin proteins (which are highly abundant in compact myelin) to create a loosely bound compartment in 1.2

Expert Opin. Drug Discov. (2015) 10(5)


PET molecular imaging of glia and myelin in drug discovery for multiple sclerosis

exchange to free tissue water. This association shortens the T2 relaxation time of bound (15 -- 40 ms) relative to either intracellular or extracellular (40 -- 200 ms) or free water [12,13]. The relative amount of the fast-relaxing component of bound water can be estimated from images acquired with different post-excitation delays to assess T2 relaxation. Validation experiments ex vivo have shown strong correlations between the bound water fraction and myelin content. Longitudinal studies in vivo have demonstrated variable recovery of myelin water content in individual lesions after an acute demyelinating episode in MS. Diffusion MRI relies on using a short-term magnetisation ‘memory’ of water protons and delaying readout of the image for a time sufficient to allow the water molecules to diffuse. The MRI is thus made sensitive to the direction of diffusion of water molecules. In bulk water (e.g., the ventricular cerebrospinal fluid), water shows high diffusion rates (high diffusivity) equally in all directions (isotropic diffusion). Water molecules associated with myelin proteins are constrained (low diffusivity). Axons and myelin restrict diffusion transverse to their axis (anisotropic diffusion). Measures of mean diffusivity increase and measures of directionality (anisotropy) decrease with demyelination, although these changes can also reflect oedema or axon loss, depending on the context [15]. The final and currently most widely used method is the magnetisation transfer (MT) imaging. This relies on difference in the resonance frequency (expressed as the chemical shift) of water molecules associated with myelin proteins and free water and the short-term magnetisation ‘memory’ of water protons. MT imaging sequences exploit the difference in chemical shift relative to bulk water to selectively transfer magnetisation to water molecules associated with macromolecules of myelin. As these macromolecule-associated water molecules exchange with free water, the overall MRI signal (which arises overwhelmingly from the free water in brain) is reduced. The greater the myelin content, the greater is the transfer of magnetisation and the greater is the decrease of the free water signal. Simply by subtracting data acquired with the magnetisation saturation pulse from that acquired without the magnetisation saturation, an image of the relative distribution of exchanging water pools can be created. Empirical correlations with post-mortem brains and preclinical studies have validated this as an index of myelin and it can be measured precisely. However, it is not yet possible to interpret it quantitatively in terms of myelin content and measuring it in a reproducible way across sites and scanners is challenging, although recent approaches promise useful approximations [14]. 2.

Molecular imaging with PET

PET signal detection PET molecular imaging involves reconstruction of an image of the tissue of interest based on the distribution of an exogenously administrated positron-emitting reporter molecule 2.1

that (ideally) interacts selectively with the target. The collision of an emitted positron with a local electron produces two photons that travel at 180 to each other. Scintillation detectors can detect these. The timing and spatial distribution of the incident photons then is used to reconstruct an image, which can be interpreted in terms of the anatomical distribution and abundance of the target. Unlike MRI or CT, the scanner itself is simply a passive signal detector. PET scanners are constructed with an array of highly sensitive scintillation detectors into which a subject can be positioned. Spatial resolution ultimately is limited by the need for positrons to travel from their origin to a local annihilation event, however. This physical limit, the practically achievable detector array densities and subject movement during scanning currently preclude an effective linear resolution greater than ~ 2 -- 4 mm. Except when the nuclide itself is used as the imaging agent (e.g., Na18F for bone studies), synthesis of a probe molecule involves radio-labelling (e.g., with 11C, 18F), a ligand that binds selectively and with high affinity (typically in the nanomolar range) to the target of interest. In some cases, an enzyme substrate is used that is selectively metabolised (or trapped) by the tissue. Given the short half-lives, low energies of PET nuclides in most common use and high PET scintillation detector sensitivity, sufficient signal-to-noise for precise measurements of signal distribution can be realised without significantly increasing long-term health risks associated with the ionising radiation. Radiosyntheses delivering highly specific activity products also allow microdoses of radioligands (typically < 10 µg) to give detectable signals, thus minimising concerns related to direct toxicities of the probe. Quantifying the PET signal The simplest measure of uptake of a radio-labelled probe in the tissue is the standardised uptake value (SUV), which is calculated as the ratio of tissue radioactivity per unit volume (at a given time and dose) divided by the injected radioactivity expressed relative to body weight (given, e.g., in units of megaBecquerel per kilogramme). Signal at any observation time must be corrected for the (known) rate of radioactive decay (a physical constant) since injection. However, SUV does not distinguish between contributions to the signal from different tissue compartments (e.g., blood, target-bound and free radioligand). More specific approaches to measuring radioligand uptake will be needed for many pharmacodynamic study applications. Conceptually, the simplest of these approaches is to normalise uptake of the radioligand in the region of interest with respect to another tissue that does not express the target, but has a comparable blood volume and similar amounts of free or non-selectively bound radioligand. As glial cells are ubiquitous in the brain, use of a reference tissue only makes sense when focal changes are being assessed. Even then, quantitative results need to be viewed critically; the choice of reference tissue inherently biases measures. 2.2


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A less-biased approach is to quantitatively model radioligand distribution between different tissue compartments (Figure 1). Such simplest model assumes only two compartments: blood and tissue. Transport rate constants between the compartments and the radioligand taken up in the tissue can be estimated using such a model. More complex models that additionally take into account, for example, free and bound radiotracer in the tissue can also be used. In some cases, models can be validated by demonstrating clinically meaningful discrimination of patients and healthy subjects or by direct comparision with an accepted ‘ground truth’ (e.g., post-mortem histology after a pre-morbid PET scan) [16]. Otherwise, the optimal choice of model must be determined empirically based on the goodness of fit of model derived parameters to empirical data. Expert opinion concerning the optimal model can also evolve over time with greater understanding of the target biology [17,18]. These considerations inevitably will slow the standardisation of methods for PET imaging of glial activation. PET radiotracer development Even so, perhaps the major limitation to the application of PET technology has been the availability of well-characterised radioligands relevant to pharmacological questions of interest. PET radioligands reported to date are described in the Molecular Imaging and Contrast Database, which is available online [19]. The development and characterisation of new radioligands often has relied on the experience of prior medicinal chemistry work for drug development using the same target. The chemical scaffolds of molecules from several drug series thus have been ‘re-purposed’ as radioligands. However, the required affinities usually are higher and requirements for selectivity of target-binding and metabolism are arguably even more demanding for ideal PET radioligands than for drug molecules. Nonetheless, the medicinal chemistry experience involved in creating a drug molecule can provide a powerful guide for PET radioligand discovery. Discovery of novel PET radioligands thus is more challenging for targets (e.g., on microglia or myelin) for which either no prior or only limited prior medicinal chemistry efforts have been made. Recent advances in approaches to design based on the application of bio-mathematical modelling after physicochemical characterisation of candidate molecules have suggested ways in which in silico methods can contribute to acceleration of medicinal chemistry for new radioligand discovery [17,20]. Microfluidics methods for PET radiochemical syntheses of candidate molecule series [21] also deserve further development and wider application as part of efforts to speed this process. 2.3

3.

Imaging microglia

Microglial activation Innate immune responses in the brain are mediated primarily by microglia and astrocytes [22]. In the absence of activating 3.1

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signals, microglia adopt a quiescent, ‘surveillance’ phenotype. Molecules released with tissue injury and activators of Tolllike receptors activate microglia. Depending on the stimulus, microglia can adapt their phenotype to effect a full range of responses extending from those that are proinflammatory to those that are immunosuppressive and locally trophic. Like microglia, astrocytes also respond to factors associated with tissue injury by adopting diverse profiles depending on the activation stimuli and context [23]. Although conventional MRI methods of T2-weighted imaging and gadolinium-enhanced T1-weighted imaging are sensitive to inflammation in the brain, there are no clinical imaging methods for independently evaluating the innate immune response that are used in routine clinical practice. MRS methods have been proposed in research applications but major questions concerning their sensitivity and specificity still need to be answered. Folate receptor imaging as a marker of macrophage activation

3.2

Two targets have been widely proposed for PET molecular imaging of activated microglia or macrophages: the folate receptor (FR) and the 18 kDa mitochondrial translocator protein (TSPO). The FR-b is a target for imaging activated macrophages [24]. The number of normal tissues expressing FR-b is limited and high expression is found in activated macrophages. The PET tracer [18F]fluoro-PEG-folate was synthesised and evaluated preclinically with both in vitro and ex vivo studies using a methylated bovine serum albumininduced arthritis model for a proof of principle [25]. More recently, application of a single photon emission tomography (SPECT) radioligand, 99m Tc EC20 (etarfolate), also binding to FR-b, was reported in a study of rat experimental autoimmune encephalomyelitis (EAE), demonstrating the potential of this approach in studies of the brain [26]. Both the PET and the related SPECT methods should be evaluated further, although the higher sensitivity and potential for more precise registration with MRI of PET (e.g., with integrated MRI-PET scanners [27]) may make this the method of choice for MS applications. TSPO as a marker of activated microglia in MS Considerable human brain imaging experience has been reported using TSPO (formerly known as the peripheral benzodiazepine receptor or PBR) as a target for evaluating microglial activation. TSPO is an 18 kDa transmembrane protein found in the outer mitochondrial membrane that plays diverse roles in cellular functions, the most well-defined being steroid synthesis. Although TSPO is expressed in many cell types, expression in healthy human brain cells is low except in activated microglia, some activated astrocytes and endothelial cells [18]. Studies of cultured glial cells and animal models suggest that whether TSPO expression is increased in both microglia and astrocytes or more selectively in microglia 3.3



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Parent plasma Cortical 2TC fit Pons 2TC fit Thalamus 2TC fit Caudate 2TC fit

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Figure 1. A. A two-compartment model is the simplest form for analyses of PET. It assumes that the radioligand is found free in the extracellular space (typically assumed in the first instance to have an equivalent concentration to the plasma-free concentration) or bound to its target in brain tissue. Data acquired during a dynamic PET scan are modelled by measuring the time course of ligand radioactivity free in plasma of arterial blood and the change in brain radioactivity reflecting blood and tissue radioligand. De-convolution of total brain time-activity curve with the arterial blood input function determines the tissue time activity curve. From these, the rate constants and an equilibrium radioligand uptake in the tissue can be defined. B. Time-activity curves differ between tissue compartments in the brain depending on both blood flow and tissue radioligandbinding. Here, an illustrative tracer with an arterial input function, modelled as the solid blue line, distributes between cortical (yellow), pons (red), thalamus (green) and caudate (blue). Time-activity curve example courtesy of Prof. Roger Gunn, Imperial College London and Imanova, Ltd. PET: Positron emission tomography.

depends on the activation stimulus [28,29]. Although to a lower level, TSPO is also expressed in lymphocytes, so these cells could also make a contribution to the brain signal in highly active MS lesions. Interpretation of the TSPO PET signal from the brain therefore depends on understanding the context [30,31]. Interpretation needs to be informed by prior studies of neuropathological material in which the cell populations contributing to TSPO expression can be characterised ex vivo. For example, increased brain TSPO radioligand signals in experimental autoimmune encephalomyelitis (EAE) and cuprizone demyelination models are associated with different neuroinflammatory pathologies. In rodent EAE, most TSPO radioligand-binding in the brain can be attributed to activated microglia, but not astrocytes [32]. By contrast, in the cuprizone model, radioligand-binding is found both in activated microglia and astrocytes in demyelinated regions [33]. Post-mortem studies of MS brains are generally consistent with findings in EAE. TSPO binding has been reported in white matter lesions, particularly at the periphery of chronic plaques, consistent with recognised distributions of activated microglia [34-36]. TSPO binding is also detected at lower levels in areas of microscopic inflammation not associated with plaques. Overall, increased TSPO binding in MS lesions is

predominantly associated with activated microglia (and macrophages in acute, active lesions) [37]. Thus, in this context, TSPO appears to provide a relatively selective marker of activated microglia and macrophages recruited into active lesions and could be used as an index of neuroinflammatory treatment response [38]. However, additional research is needed to characterise the relationship between microglial phenotypes (e.g., bias towards proinflammatory or immunosuppressive cytokine expression) [22] to evaluate the potential clinical significance of any pharmacodynamic effects observed. TSPO-targeted radioligands and applications in MS

3.3.1

Many PET radioligands have been developed that show selective, reversible binding for TSPO. The most extensively studied of these is [11C]PK11195 [39]. However, PET studies using this tracer are believed to be limited by the radioligand’s modest binding affinity, higher non-selective binding and relatively poor extraction by the brain [17]. There has, therefore, been considerable interest in developing new TSPO radioligands with high affinity binding for TSPO, lower non-selective binding than [11C]PK11195 and better pharmacokinetics. Approximately 50 alternative radioligand


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candidates have been described [40]. However, only a handful, including [11C]DAA1106 [41], [18F]PBR111 [42], [11C] PBR28 [43] and [18F]FEPPA [44], have been evaluated in humans. The highly selective binding of these ‘second generation’ TSPO radioligands have been confirmed in vivo by blocking of TSPO with unlabeled drugs in animals [43,45] and humans [46]. They differ in affinity for the target, differences in relative binding affinity for people carrying the alternative TSPO alleles (see below) and possibly also in their non-displaceable (and therefore non-selective) binding fraction in the brain. Nonetheless, these radioligands all bind to the same or overlapping sites on TSPO as PK11195, as they exhibit mutually competitive binding [35]. It, therefore, should be possible to interpret their selective binding in similar ways. Comparative studies to confirm this are needed. [11C]PK11195 has been the most widely used TSPOtargeted PET radioligand to date. Modelling methods for quantitative estimations of total volume of distribution (VT) are well described [47,48], but their validation is difficult and efforts have been limited. Evaluation of TSPO expression based on tissue uptake of the newer, second-generation radioligands demands knowledge of subject genotype: carriers of the lower frequency rs6971polymorphism in the human TSPO gene (more common particularly amongst Northern European Caucasians) have a form of the protein with substantially reduced affinity for most of the second-generation radioligands [17,49,50]. Application of TSPO PET to evaluate microglial activation in MS is supported by associations observed in clinical studies. In people with MS, TSPO PET shows highest radioligand uptake in gadolinium-enhancing lesions, consistent with the expected focal infiltration of activated microglia and macrophages (Figure 2) [51,52]. A recent pilot study provides new data showing that microglial activation in normal-appearing white matter of people with a clinically isolated syndrome is higher for those who develop clinical MS within 2 years, suggesting an association with severe disease and greater inflammatory neuropathology [53]. Other studies have shown higher signal in people with secondary progressive MS than relapsing remitting MS, consistent with the expected higher inflammatory lesion burden with longer disease duration in the former group [54,55]. The TSPO uptake in white matter seems to explain much of the disability observed in a crosssectional cohort (Figure 3). Additional validation has come with the study of lesions in grey matter (GM), an area of particular interest, as these lesions are relatively poorly defined by MRI. They are characterised neuropathologically by pronounced macrophage and microglial activation in their earlier stages of evolution, although lesions become relatively acellular later [56]. Increased signal particularly in subcortical GM of the thalamus was an early observation made using [11C]PK11195 TSPO PET [34]. Recent work has shown that neocortical GM TSPO radiotracer uptake correlates with both expanded disability status scale and 6

MS impact scale-29, suggesting that TSPO PET can provide an estimation of the contribution of GM inflammatory activity relevant to disability in MS [54]. A recent, pragmatic pilot longitudinal study of MS patients explored pharmacodynamic effects of glatiramer acetate and reported decreases in brain [11C]PK11195 uptake after 1 year of treatment [38]. These studies together highlight the potential utility of TSPO PET in the development or evaluation of MS immunotherapy. The PET TSPO signal reports on chronic inflammatory changes that are not detected using conventional MRI methods. It non-invasively defines disease-relevant neuropathological heterogeneity within T2 lesions [55,57] and may be able to detect GM inflammation not otherwise able to be characterised by MRI [54]. TSPO PET is also sensitive to microglial activation in white matter defined non-specifically by its abnormal magnetisation transfer signal [55]. However, whereas a proportion of the signal is molecularly selective for TSPO, all of the ligands will show non-selective binding. Challenges to applications of TSPO radioligands in clinical studies of MS

3.3.2

There are several questions that need to be answered if TSPO PET imaging is to become practical and informative as an end point in drug development. First, do the new, ‘second generation’ TSPO PET radioligands bring practical benefits relative to [11C]PK11195? Answering this important question directly is difficult because direct comparisons have not been performed. In addition, quantitative evaluations based on the earlier studies with second-generation agents [58] are confounded because they were conducted before recognition of the impact of the rs6971 polymorphism population variation on radioligand-binding and thus report very imprecise measures ([11C]PBR28, e.g., shows a 50% reduction in TSPO selective binding in people carrying a single allele with the low frequency rs6971 polymorphism) [50]. Despite this, the results of these studies are generally consistent with -- and extend -- the findings using [11C]PK11195. [11C]vinpocetine BPND was higher regionally and globally, with the highest signal around plaques, a region where neuropathological studies show higher microglial and macrophage density in chronic lesions [59]. Similar results were reported using [18F]PBR111 in a separate cohort [55]. Quantitative comparisons of relative signal change in clinical studies using similar patient populations suggest that signal-to-background is modestly improved with second-generation PET radioligands relative to [11C]PK11195 (David Owen, Imperial College London and PMM, unpublished observations). Autoradiography ex vivo highlights this [35]. We cautiously conclude that use of one of the second-generation agents should improve the estimates of VT relative to what has been possible with [11C] PK11195. A second problem that needs further evaluation is the test--retest variance in healthy subjects and in people with MS. This depends on both biological and technical factors. The latter includes, for example, the precision of the input



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Figure 2. Illustrative single subject brain scans showing brain microglial activation in a person with secondary progressive multiple sclerosis as assessed using the TSPO PET radioligand [11C]PBR111. On the left is a T2-weighted MRI scan acquired on the same day as the PET image overlaid on the white matter volume to the right. This highlights that microglial activation is heterogeneous across lesions even in the same subject. Higher uptake is shown as yellow on a red-yellow colour scale. Images courtesy of Dr Alessandro Colassanti, Imperial College London. PET: Positron emission tomography; TSPO: Translocator protein.

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Figure 3. The relationship of disability to a measure of brain microglial activation assessed as the relative white matter radioligand uptake (volume of distribution) across a group of people with multiple sclerosis and different levels of disability (assessed using MSSS) is shown. Further details are described in [55]. MSSS: Multiple Sclerosis Severity Score; TSPO: Translocator protein.

function or reference region signal used either for modelling the tissue uptake or calculating the relative tissue uptake. Little reproducibility data have been reported. At best,

test--retest reproducibility may be 5 -- 10% with the use of tissue reference models, but it appears to be much higher with full modelling of VT [55,60]. Although the apparently high variance in VT may reflect the challenge of estimating an input function from blood measures, given the very low free fraction of these ligands (because of TSPO radioligand binding to blood components including platelets and white blood cells [17]), it could be a consequence of variable biological factors. A large biological contribution to variance may not be unexpected; systemic expression of inflammatory mediators can be highly variable even in healthy subjects [61] and changes in systemic inflammatory state can be reflected rapidly as brain microglial activation [29]. This question needs further research. Another question limiting interpretation is the lack of understanding of the range of microglial activation phenotypes that are associated with increased TSPO expression. Arguably the most important question to be addressed for applications in drug development is whether increased signal from current TSPO-targeted radioligands reflects increased proinflammatory or both proinflammatory and immunosuppressive [62], microglia. Finally, there is the question of the accuracy of TSPO PET as a measure of inflammatory microglial TSPO expression. One aspect of this is defining the cells in the brain contributing to the high levels of TSPO being measured. At present, we can only address this directly through independently conducted ex vivo binding studies with pathological material. Accuracy also depends on the proportion of the TSPO radioligand that is displaceable from the target by competing ligand (reflecting binding to a specific target site) relative to the radioligand that is not displaceable (i.e., non-saturable binding). In general, this will vary between radioligands and can only be well estimated in vivo in the tissue of interest. A recent report illustrated a practical approach to characterising this by an in vivo competition study with the unlabeled TSPO ligand XBD173, which competes for the same binding site as current TSPO radioligands and can be safely administered to humans at pharmacological doses [46]. This study concluded that in the brains of healthy subjects, ~ 60% of the [11C] PBR28 uptake in healthy human brain is displaceable (and therefore represents selective binding to TSPO) [46]. We expect that the proportion of selective binding will be higher with an active inflammatory process; this methodologically important issue needs direct study. 4.

Molecular imaging of astrocyte activation

Methods for PET molecular imaging of activated astrocytes are much less well developed than the methods used for microglial imaging. Any early application of these methods as pharmacodynamic measures therefore should be made in conjunction with independent efforts to validate the measures. For example, interpretation of results will demand correlative data to evaluate the magnitude of signal change that is


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clinically or pharmacologically meaningful, the dynamic range of the measure and its test--retest variability. As noted above, under some conditions, TSPO binding reflects astroglial activation (or transformation in malignant gliomas [63]). Other radioligands described below are becoming available that promise to distinguish astrocytic from microglial activation. [11C]acetate uptake as a measure of astrocyte metabolism


4.1

As noted above, because astrocytes express high levels of the MCT relative to other glial cells or neurons, they preferentially metabolise acetate. A recent small pilot study demonstrated greater [11C]acetate uptake in brains of people with MS than in healthy volunteers, consistent with higher densities of activated astrocytes in the former group [64]. The relative increase was more prominent in white matter and was proportional to the number of lesions identified by MRI. Although of interest, this report should be viewed only as a proof of principle. It highlights first steps towards realising the potential to re-purpose a radiotracer already used for some time for other applications (e.g., studies of cardiac metabolism). An obvious limitation to the use of a metabolic rate as a pharmacodynamic measure is that the therapeutic concept needs to be framed in terms of similar metabolic parameters. Monoamine oxidase B is a marker of astrocytes Other molecularly selective targets for imaging the activated astrocytes have also been suggested. In the brain, the enzyme monoamine oxidase B (MAO-B) is localised predominantly in astrocytes, where it is localised to the outer mitochondrial membrane. Both [11C]L-deprenyl and [11C]deuterium-Ldeprenyl ([11C]DED) bind to MAO-B with high affinity and selectivity (at tracer doses) [65,66]. The latter radioligand has more favourable pharmacokinetic properties. Consistent with increased astroglial density in the brain with aging, a previous PET study with [11C]DED shows that PET signal is increased in healthy older individuals. Clinical research applications have included studies of epilepsy and Creutzfeldt--Jakob disease [65,67,68]. In people with temporal lobe epilepsy, increased [11C]DED binding is lateralised with the ictal focus, providing an indirect validation of the radioligand in human applications. However, metabolites of L-deprenyl and DED also bind to MAO-B and complicate quantitative interpretation of the time--activity relationships on which estimates of VT are based. To address the latter confounds, [11C] SL2511.88 was developed as an alternative MAO-B selective tracer [69]. In a small methods validation study, the potential for human use was demonstrated and preliminary assessments of test--retest variation and selectivity were made [70]. It is encouraging that most of the total distribution volume could be attributed to MAO-B activity and that the coefficient of variance for repeat measures was moderately low (£ 10%). However, to 4.2

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our knowledge, it has not been evaluated with MS (or any other disease) yet. This, therefore, should be viewed as a promising radioligand in the earliest stages of evaluation as a pharmacodynamic marker. The I2-imidazoline receptor provides an additional PET imaging target selective for astrocytes

4.3

Although at an even earlier stage of development, another PET molecular imaging probe has been proposed that currently is being validated for human use. Like MAO-B, the I2-imidazoline receptor is expressed relatively selectively on astrocyte mitochondrial membranes and upregulation may play a fundamental role in the biology of reactive gliosis [71]. Changes in tissue expression of the receptor are associated with increased brain astrocyte activation in a range of diseases, including depression and addiction, as well as in neurodegenerative disorders such as Alzheimer’s disease and Huntington’s disease. The radioligand [11C] (2-(4,5-dihydro-1H-imidazol2-yl)-1- methyl-1H-indole) ([11C]BU99008) has high affinity for the receptor and is blood--brain barrier penetrant in rodents [71], pigs [72] and monkeys [73], in which it shows a heterogeneous uptake between brain regions consistent with the known distribution of expression of the I2-imidazoline receptor. Evaluation of its potential as a human PET radioligand is ongoing and promising (Professor D Nutt, Imperial College London, personal communication). This radioligand is not yet ready to be used as a pharmacodynamic measure but is one to watch. 5.

Molecular imaging of brain myelin

Myelin in the brain and spinal cord is the primary immune injury target in MS. Myelin loss thus reflects a core feature of the neuropathology and provides an index of the burden of disease. Additional direct and quantitative myelin imaging methods are needed to support the development of new drugs to enhance remyelination [74]. Molecules based on the histological stain Congo Red that bind brain amyloid have also shown binding to myelin. The first widely used amyloid radioligand, N-Methyl-[11C]-2-(4¢methylaminophenyl)-6-hydroxybenzothiazole (Pittsburgh Compound B or PIB), binds to white matter and amyloid [75]. PIB binding to white matter distinguishes demyelinated from myelinated regions in preclinical models and defines differences consistent with the patterns of relative white matter demyelination in people with MS apparent in paired MRI scans [76]. Interpretation of the PIB signal is not expected to be confounded by significant amyloid deposition in most applications to MS, as amyloid deposition is not a feature of its pathology. PIB PET, therefore, could build on the considerable experience that molecular imaging sites have in its use for Alzheimer’s disease studies to enable PET evaluation of myelin in MS.




A.

B.

Pre-EAE

EAE

Figure 4. A. Three-dimensional PET and CT fusion images showing the whole spinal cord in a Sprague Dawley rat 42 days after immunisation with immunogenic MOG, which induces EAE. The spatially heterogeneous, green colour highlights the loss of myelin signal with induction of EAE as assessed using the myelin-selective PET radioligand [11C] MeDAS. B. Sagittal and coronal PET images showing decreased uptake of [11C]MeDAS in the lower thoracic spinal cord after EAE immunisation relative to before (pre-EAE) immunisation. Images courtesy of Dr Yanming Wang, Case Western Reserve University. EAE: Experimental allergic encephalomyelitis; MOG: Myelin oligodendrocytic glycoprotein; PET: Positron emission tomography.

However, the major potential advantage of PET molecular imaging of myelin over well-established, less-expensive nonconventional MRI lies in the potential to provide an absolute, quantitative measure. In clinical applications for Alzheimer’s disease, the PIB PET signal is interpreted qualitatively. Whereas relative binding to myelin can be assessed, quantitative binding may not be able to be well interpreted as an absolute measure, as PIB binding to myelin is neither saturable nor displaceable [75]. The myelin binding site(s) for PIB also has not been characterised. Finally, there appear to be illunderstood influences on PIB binding to myelin that may confound interpretation. In rodents, [11C]PIB uptake across the brain and cerebellum does not correspond with myelin distribution [77]. In principle, alternative PET radioligands that appear to be highly selective for myelin and which show the saturable binding needed for an absolute, quantitative measure of myelin content will be more accurate and precise [78]. [11C] N-methyl-4,4¢-diaminostilbene ([11C]MeDAS), has shown pharmacokinetics, tissue binding and blood--brain penetration characteristics favourable for imaging [78]. The highest binding in the brain is in white matter. MeDAS also shows high affinity binding that is displaceable and is blocked by an antibody

to myelin basic protein, which has b-sheet secondary structures, to which this class of molecules shows high affinity. Consistent with this preliminary characterisation, [11C] MeDAS PET signal decreases with inflammatory demyelination in EAE [79]. The utility of [11C]MeDAS PET for longitudinally assessing myelin content has been evaluated in the spinal cord in vivo using a focal rat demyelination model based on stereotactic injection of lysophosphatidycholine (LPC). [11C]MeDAS uptake was lowest when demyelination was at its maximum and gradually increased with later spontaneous remyelination between after injection, tracking the time course of histochemical measures of myelin in post-mortem tissue and clinical symptoms. The potential of [11C]MeDAS PET to monitor pharmacodynamic effects of a potential myelin repair therapy was demonstrated with a study of hepatocyte growth factor after this LPC demyelination (Figure 4). These imaging results were validated by double-blinded immunohistochemical observations of post-mortem tissue sections harvested at each time point. The feasibility of its use in humans has yet to be demonstrated, but, preclinically, [11C]MeDAS currently is a preferred PET tracer to monitor myelin changes in the brain and spinal cord in vivo [77]. 6.

Conclusion

Microglial imaging using PET can be performed using both 11 C and 18F radioligands. Although there is some promise that FR imaging can be used for imaging macrophages in the brain, the only demonstrations of the potential of PET microglial imaging for clinical studies in MS to date have employed radioligands targeting the 18 kDa TSPO that is upregulated in both macrophages and activated microglia. However, because TSPO is also expressed in vascular endothelial cells and is increased in the astrocytes activated with some neuroinflammatory processes, the TSPO PET signal needs to be interpreted in the context of what is known about the underlying neuropathology. In applications to MS, it is clear that, as expected, the TSPO PET measures complement, rather than simply reflect, measures of immune response derived from conventional MRI. The relatively low signal variance of TSPO PET measures based on a reference tissue model (5 -- 10%) supports the case for using TSPO PET for monitoring pharmacodynamic effects on focal (or multifocal) inflammatory processes, but the lack of understanding of factors contributing to variance in the VT is a concern. Further work is needed to interpret the signal in terms of microglial activation phenotype and to better understand potential sources of biological variance. PET molecular imaging of astrocyte activation for human pharmacological studies based on radioligands targeting either the MAO-B ([11C]DED) or the I2-imidazoline receptor ([11C]BU99008) is possible but needs further evaluation to understand signal selectivity and to enable interpretation across different pathologies quantitatively and reproducibility. These agents appear likely to be more practical to use as


9



astrocyte activation markers than changes in [11C]acetate metabolic kinetics, although this metabolic radiotracer already is well characterised for human use in other applications. Despite the currently high drug development need, PET myelin imaging is not yet ready for application as a decision-making tool in drug development studies, although the groundwork has been laid for possible rapid progress. A displaceable radioligand with a known molecular target on myelin is important if quantitation and interpretation is to be made confidently, particularly in applications in which dynamic changes in myelin content and structure are expected. [11C]MeDAS may be the most promising preclinical radioligand, but it needs toxicological evaluation and first demonstration of use in humans. Precise spatial localisation of the signal and imaging resolution may be particularly important for myelin imaging in MS, in which focal demyelination is variable and localised to lesions of heterogeneous size and shape. Development of improved methods for co-registration of MRI and PET scans and motion correction of PET scans will help in localisation of the signal. These problems may become more tractable to address as integrated MRI-PET scanners begin to be used more widely for brain studies. 7.

Expert opinion

The combination of sensitivity and selectivity makes PET molecular imaging attractive as a pharmacodynamic assessment tool in therapeutics development for MS (and other neurological diseases). A number of PET radioligands hold promise as biomarkers for patient stratification or pharmacodynamic end point assessment in applications, but major challenges remain to be addressed if the methods are to be practical for any but ‘niche’ applications. The molecular selectivity of the PET radioligands discussed here illustrates well both the major strength and a potential weakness of using molecular imaging in this way. The selectivity of the measures contribute to testing well-posed hypotheses, but the small number of available radioligands limits the questions that can be tested. Also, despite proofs of principle, all of the novel PET radioligands discussed here need further validation in the specific context of the desired use before confident application in drug development (e.g., by employing a translational pathway using the same biomarkers in preparatory preclinical studies [80]). As reviewed earlier in this expert opinion, TSPO PET radioligands progressed the farthest towards becoming a useful tool. The TPSO signal could be used even now as an exploratory complement to conventional MRI measures for stratifying patients by the extent of inflammatory pathology or as a pharmacodynamic marker in applications focusing on innate immune responses, but three major problems need to be addressed before they can be used with greater confidence. First, factors contributing to population variance and reproducibility of the signal need to be understood. What biological factors contribute to variance of modelled signal that 10

should be controlled for optimal assessments? In conjunction with this, modelling methods for quantitative analyses need to be validated and harmonised between research centres. Finally, the biological interpretation of the signal needs to be clarified. Is TSPO binding similarly increased in activated proinflammatory to immunosuppressive-activated microglial phenotypes? Even with this, there will be some uncertainty about the cell population contributing to any TSPO signal change given the potential for expression from macrophages and activated astrocytes, as well as active microglia in MS. FR PET imaging could provide a way of differentiating drug effects on activated microglia from those that only modulate infiltrating macrophages in the future, but a human proof of principle has yet to be reported. This approach is still in the earliest stages of development as a biomarker. PET methods to facilitate testing of clinical drug development hypotheses regarding astrocyte activation in humans are more advanced, but they are still in need of further characterisation. The recent proof of principle for evaluation of astrocyte activation by [11C]acetate uptake allows ‘re-purposing’ of a radiotracer that has been used for some time in many PET centres. The underlying metabolic principle is well established in neurochemistry and well suited to preclinical evaluation as part of a drug development pathway. Both MAO-B PET and I2-imidazoline PET radioligand may provide future alternative activated astrocyte PET imaging biomarkers for other early drug development applications. By analogy with what was done for MRI measures early after their introduction [81,82], to use these new PET biomarkers, careful validations now need to be performed that correlate neuropathological variations with radioligand binding to these targets in brain tissue from people with MS post-mortem. The potential of PET imaging biomarkers of brain myelin needs to be considered in the context of well-established, albeit indirect, MRI measures of relative brain myelin content. Our opinion is that future molecular imaging of brain myelin with PET should be viewed as an emerging complement rather than as an alternative to these. A quantitative measure of brain myelin could be used to ‘calibrate’ the relative MRI measures [4]. We are optimistic that [11C]MeDAS, which binds to myelin with higher affinity at a saturable site, can be translated for human use in the future; successful translation cannot be assumed because of the potential for confounding differences in metabolism and pharmacokinetics between rodents and humans. Another consideration is that, to be useful in the evaluation of remyelinating therapies in MS in which demyelinated lesions defined on MRI can be small, current approaches to spatial registration of the PET signal and accompanying MRI need improved: misregistrations on the order of even a few millimetres in a linear dimension would have a significant impact on signal averaged from a smaller demyelinating lesion volume. Encouragingly, though, there are emerging opportunities to substantially




improve this by acquisition of intrinsically co-registered data from integrated MRI-PET scanners [27]. Although not essential for small proof-of-principle studies, applications in PET for drug development generally would be enabled by a potential for multi-centre studies. None of the PET radioligands described in this review are available for use outside of specialised facilities able to manufacture them on site because the short half-life of the [11C] radioligands precludes central manufacture and distribution and the [18F] radioligands are not approved for any indication or available commercially. In the absence of radiopharmaceutical manufacturing at scale, costs of the types of PET scans discussed are also high as much as an order of magnitude greater than for MRI. Use of PET in multi-centre trials also demands standardisation of quality control, acquisitions, reconstruction and modelling, just as for MRI [83]. All of these issues could be addressed, but the rationale for doing so needs to be stronger; ways of addressing them are individually precedented by experience in applications of fluorodeoxyglucose PET [84]. Our opinion is that PET molecular imaging holds promise for new drug development in MS, particularly with greater demands for differentiation of new drugs being made by regulators and payers. To realise this promise, greater research investment in the area is needed. We believe that there is a case to make this commitment, given the size of the MS therapeutics market and the amount of drug development activity in it (and related areas). PET molecular imaging studies already have shown that they can extend understanding of aspects of the pathological heterogeneity of MS lesions not reported by MRI [34,54,55]. An important research direction lies in relating this heterogeneity to clinically meaningful outcomes, so that the methods can contribute to more accurate predictions of prognosis and for evaluating the efficacy of a Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Affiliation

Paul M Matthews†1 & Gourab Datta2 † Author for correspondence 1 Professor, Imperial College London, Division of Brain Sciences, Department of Medicine, E515, Burlington Danes Building, Du Cane Road, W12 0NN London, UK Tel: +44 02075942612; Fax: +44 02075946548 ; E-mail: [email protected] 2 Imperial College London, Division of Brain Sciences, Department of Medicine, London, UK

Lipid composition of myelin in multiple sclerosis.

Drug therapy for multiple sclerosis.

Multiple molecular forms of glia maturation factor.

Age independently affects myelin integrity as detected by magnetization transfer magnetic resonance imaging in multiple sclerosis.

Brain and cord myelin water imaging: a progressive multiple sclerosis biomarker.

Myelin recovery in multiple sclerosis: the challenge of remyelination.

Identity of myelin basic protein from multiple sclerosis and human control brains: discovery of a genetic variant.

Multiple sclerosis: Drug-enhanced remyelination in a multiple sclerosis model.

Myelin autoreactivity in multiple sclerosis: recognition of myelin basic protein in the context of HLA-DR2 products by T lymphocytes of multiple-sclerosis patients and healthy donors.

Pioglitazone regulates myelin phagocytosis and multiple sclerosis monocytes.

Multiple sclerosis and cell-mediated hypersensitivity to myelin A1 protein.

Quantified CSF antibody reactivity against myelin in multiple sclerosis.

Immune response to myelin basic protein in multiple sclerosis.

Imaging phenotypes in multiple sclerosis.

PET imaging in multiple sclerosis.

Computerised axial tomography in multiple sclerosis.

Imaging in multiple sclerosis and related disorders.

Systems Pharmacology in Small Molecular Drug Discovery.

Drug Discovery by Molecular Imaging and Monitoring Therapy Response in Lymphoma.

Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis.

The Latest Innovations in the Drug Pipeline for Multiple Sclerosis.

Magnetic resonance imaging of myelin using ultrashort Echo time (UTE) pulse sequences: Phantom, specimen, volunteer and multiple sclerosis patient studies.

Imaging Markers for Monitoring Disease Activity in Multiple Sclerosis.

Role of Molecular Dynamics and Related Methods in Drug Discovery.