Identification of AV-1451 as a Weak, Nonselective Inhibitor of

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Identification of AV-1451 as a Weak, Nonselective Inhibitor of Monoamine Oxidase Lindsey R Drake, Jonathan M Pham, Timothy J Desmond, Andrew V. Mossine, So Jeong Lee, Michael R. Kilbourn, Robert A Koeppe, Allen F Brooks, and Peter J. H. Scott ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00326 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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ACS Chemical Neuroscience

Identification of AV-1451 as a Weak, Nonselective Inhibitor of Monoamine Oxidase Lindsey R. Drake,a† Jonathan M. Pham,b† Timothy J. Desmond,b Andrew V. Mossine,b So Jeong Lee,b Michael R. Kilbourn,b Robert A. Koeppe,b Allen F. Brooks,b and Peter J. H. Scotta,b* a

Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. b

Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA.

Abstract [18F]AV-1451 is one of the most widely used radiotracers for positron emission tomography (PET) imaging of tau protein aggregates in neurodegenerative disorders. While the radiotracer binds with high affinity to tau neurofibrillary tangles, extensive clinical studies have simultaneously revealed off-target tracer accumulation in areas of low tau burden such as the basal ganglia and choroid plexus. Though there are a number of possible reasons for this accumulation, it is often attributed to off-target binding to monoamine oxidase (MAO). In this paper, we investigate the association between [18F]AV-1451 and MAO through: i) enzyme inhibition assays; ii) autoradiography with post-mortem tissue samples, and iii) nonhuman primate PET imaging. We confirm that [18F]AV1451 is a weak inhibitor of MAO-A and -B, and that MAO inhibitors can alter binding of [18F]AV1451 in autoradiography and in vivo PET imaging. Keywords: positron emission tomography (PET) imaging; tau PET; Alzheimer’s disease; monoamine oxidase (MAO); flortaucipir. 1. Introduction Alzheimer’s disease (AD) is a progressive and ultimately fatal neurodegenerative disorder that is currently without a cure. Developing a treatment for AD is thus a priority research area involving a multipronged approach that includes: (i) efforts to understand disease pathophysiology, (ii)

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identification of imaging biomarkers that can be used to develop diagnostic tests (e.g. molecular imaging) for staging disease, enriching clinical trials, and monitoring patient response to disease modifying therapies,1, 2 and (iii) exploitation of these advances to develop effective therapeutics. Numerous targets are being considered, with the most aggressive research efforts targeting extracellular amyloid-beta (Aβ) plaques. However, to date strategies that clear Aβ from the AD brain in an attempt to ameliorate cognitive impairment have not been successful. In response, there has been a shift to targeting intracellular tau neurofibrillary tangles (NFTs),3 composed of hyperphosphorylated tau, as a point of therapeutic intervention since NFT burden correlates with cognitive decline and Braak staging.4 To support these efforts, several positron emission tomography (PET) radiotracers targeting tau NFTs have been developed (for recent reviews, see:59)

with [18F]AV-1451 (Flortaucipir, T807, 1)10 being one of the most widely used in clinical studies.

Independent validation efforts from several groups have confirmed localization of [18F]AV-1451 with tau NFTs, visualized by immunological methods.11,

12

However, tau PET has proven

challenging13 because of complicating factors such as binding of first-generation PET radiotracers like [18F]AV-1451 to other targets not yet identified in similar immunological studies.11, 14, 15 This off-target binding appears to be reflect actual binding to a target other than tau, based on slower tracer clearance compared to the reference region, cerebellar gray matter. The nature of the offtarget binding of [18F]AV-1451 is not fully understood, but it has been attributed to a variety of targets other than tau NFTs, including pigmented cells in the CNS, elevated iron levels, and/or monoamine oxidase (MAO) A and/or B.16-21 However, neuromelanin-containing cells, chiefly in the substantia nigra, are known to contain high amounts of MAO,22 and ferroptosis (a type of programmed cell death dependent on iron) is associated with neuroinflammation and astrocytes which express both MAO-A and -B.23 Therefore, it is possible that the off-target signal is only (or predominantly) due to MAO, and in vitro studies have revealed high-affinity interactions between [18F]AV-1451 and MAO.24, 25 For example, Vermeiren showed that AV-1451 binds to recombinant human MAO-A with a Kd value of 1.6 ± 0.4 nM and inhibits human enzyme activity with a potency of 1 µM. Similar results were obtained for MAO-B (Kd = 21 ± 9 nM, inhibition at 10 µM).12 With regards to MAO, it is not yet fully understood what effects, if any, off-target binding could have on human imaging with [18F]AV-1451. It has been shown that MAO-B is more highly


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abundant in the brain,26 but Hansen and coworkers retrospectively analyzed a small cohort of patients taking MAO-B inhibitors and found no effect on [18F]AV-1451 uptake, binding or retention.27 However, these patients were determined to be tau negative and the length of time that patients had been taking MAO inhibitors (MAOIs) was unknown. Smith et al. compared [18F]AV1451 retention in study participants treated with the MAO-B inhibitor rasagiline to those who did not receive the medication and also observed no difference in AV-1451 retention in the cerebral cortex, globus pallidus, or substantia nigra.28 They obtained similar results using SUV and SUVR values, to account for potential binding to MAO-B expression in the cerebellum reference region. However, like Hansen’s study, the tau burden in subjects taking rasagiline, as well as prescribing information (dose and duration of treatment) for the MAOI, were not discussed. This differs from results obtained by Ng, who demonstrated reduction in binding and retention of the tau PET tracer [18F]THK5351 upon administration of selegiline,29 and suggests that AV-1451 perhaps has lower affinity for MAO-B than THK5351. This ongoing discussion around the off-target binding of [18F]AV-1451 encouraged us to continue our investigations with the radiotracer, and we herein report pre-clinical evaluation of [18F]AV1451 in regards to its association with MAO using: i) inhibition assays with MAO-A and MAOB, ii) autoradiography and immunohistochemistry studies with post-mortem human brain tissue sections, and iii) blocking studies with reversible MAO inhibitors performed in healthy non-human primates to investigate any influence on [18F]AV-1451 binding and retention in vivo. 2. Results Clinical Imaging Clinical imaging with [18F]AV-1451 revealed presumed off-target radiotracer accumulation in the substantia nigra (SN), basal ganglia (BG) and choroid plexus (ChP) in both cognitively normal subjects (Figure 1A) and AD patients (Figure 1B). AD patients also show the expected tau signal in, for example, entorhinal cortex (EC), lateral temporal lobe (TL) and occipital cortex (OC) (Figure 1B).



Figure 1. Two slices of representative clinical [18F]AV-1451 PET scans of a 69 year old normal control (A) and 71 year old tau-positive AD patient (B). Both sets of images clearly show offtarget binding in basal ganglia (BG), choroid plexus (CP) and substantia nigra (SN). AD patient also has significant tau burden in entorhinal cortex (EC), lateral temporal lobe (TL) and occipital cortex (OC). PET images are mean SUVR images 75-105 min post-injection; MRI images are MP RAGE (T1 weighted) images.


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MAO-A and MAO-B inhibition assays MAO-A and MAO-B inhibition assays revealed that AV-1451 is a weak, nonselective inhibitor of both enzymes (MAO-A: Ki = 25.00 ± 1.19 μM, R2 = 0.8685, n = 6; MAO-B: Ki = 28.37 ± 1.22 μM, R2 = 0.8236, n = 6). Autoradiography Studies [18F]AV-1451 MAO-B binding was evaluated on postmortem human brain tissue sections in the presence of the irreversible MAO-B inhibitor deprenyl. Substantia nigra (SN) and cerebellum (CBL) sections were used in this study because of the nonspecific binding observed in the SN during human [18F]AV-1451 PET scans (Figure 1) and the use of CBL as a reference region for quantitation.30 Specific binding of [18F]AV-1451 to tissue was determined by subtraction of nonspecific binding (identified with excess [19F]AV-1451) from total binding. Specific binding was approximately 400 times higher in diseased (AD and dementia with Lewy bodies (DLB)) SN sections when compared to age-matched control SN sections; this increase is consistent to a much lesser extent in CBL. Total binding of [18F]AV-1451 was significantly diminished in competition with deprenyl in both SN and CBL sections (Figure 2). There was no evidence of a dose-response relationship between [18F]AV-1451 binding and the two deprenyl doses tested. For example, there was no significant difference between [18F]AV-1451 binding in the presence of low and high concentrations of deprenyl in the cerebellum (p = 0.4743). Interestingly, in substantia nigra the low concentration of deprenyl displaced more AV-1451 than the high concentration (p = 0.0037). We do not have an explanation for this finding, other than that the higher concentration of deprenyl (500 µM) may have been too high for such an experiment although we did not observe any precipitation. Tissue sections for both SN and CBL from cognitively normal subjects did not have statistically significant differences in [18F]AV-1451 total binding when compared to nonspecific or MAO-B inhibition challenges (see Supporting Information).



Figure 2. [18F]AV-1451 binding to A: diseased substantia nigra (SN) and B: diseased cerebellum (CBL) tissue sections. Binding is normalized to section area and individual experimental dose. Specific binding was calculated as the difference between nonspecific and total binding. Averages are shown as percent of total binding, ± standard error of the mean. Low deprenyl challenge was 500 nM, high deprenyl was excess (500 µM). Significance was calculated using Tukey’s multiple comparisons test. Immunohistochemistry Immunohistochemistry (IHC) was performed on postmortem tissue sections to determine the relationship between [18F]AV-1451 binding, MAO-B expression and tau load (see Supporting Information for more information including representative IHC images). Since we do not fix tissue for autoradiography studies we could not use the same sections for immunohistochemistry and instead used adjacent tissue sections to those used for autoradiography. Compared to sections from age-matched controls, MAO-B immunoreactivity was 3-fold higher in diseased SN sections and 2-fold higher in diseased CBL sections (Figure 3). Tau burden in diseased SN and CBL is known to be low and, while immunohistochemistry identified some tau in both areas, it was negligible compared to MAO (Figure 3). In the diseased brain tissue samples where substantia nigra and cerebellum tissue from the same patient were available, MAO-B immunoreactivity was found to be ~4 times higher in the substantia nigra compared to the cerebellum, while anti-tau reactivity was higher by a factor of ~24. Pearson correlation tests were performed to compare MAO-B or tau immunoreactivity and [18F]AV-1451 binding (see Supporting Information). The only significant


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correlation found was MAO-B immunoreactivity and [18F]AV-1451 total binding in diseased SN tissue sections (r = 0.9592, p = 0.0408).

Figure 3. Immunoreactivity on postmortem human brain tissue sections. SN (top) and CBL (bottom) sections comparing diseased brain tissue to cognitive normal control for MAO-B and tau immunoreactivity. Nonhuman Primate Imaging and Blocking Studies In nonhuman primate brain, blocking with the reversible MAO-A inhibitor moclobemide resulted in a modest increase in SUVpeak in the cerebellum and basal ganglia, while pretreatment with the reversible MAO-B inhibitor lazabemide produced minor increases in the SUVpeak of the two regions (see Supporting Information for full time-radioactivity curves). SUV values in the final four frames of imaging (approximately 55, 65, 75, and 85 minutes post-injection) for various regions-of-interest (ROIs) were averaged (Figure 4A) and clearly show a reduction in [18F]AV1451 uptake following treatment with moclobemide or lazabemide. SUVs divided by the averaged cerebellar-SUV provided normalized SUVRs (Figure 4B), and the apparent trend for SUV was altered because of the presence of displaceable off-target binding in cerebellum. For example, following pretreatment with lazabemide, the normalized value in the basal ganglia increased



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slightly, while a decrease in the normalized SUV was observed following blocking with moclobemide (Figure 4B). 3. Discussion We have been using [18F]AV-1451 for clinical imaging in a number of research studies since 2017 (Figure 1). These imaging studies have revealed presumed off-target radiotracer accumulation in the substantia nigra, basal ganglia and choroid plexus in both cognitively normal subjects (Figure 1A) and AD patients (Figure 1B), consistent with literature reports (vide supra). Our investigation into the origin of [18F]AV-1451 off-target binding was sparked by these clinical findings, as well as ongoing debate in the tau-PET community concerning the origins of this off-target PET signal. Although an in vitro screen reported during the initial development of [18F]AV-1451 did not identify any interactions that warranted a toxicity concern,10 several groups, including our own, have subsequently identified MAO as a potential off-target binding partner for [18F]AV-1451.25, 31, 32

Off-target binding to MAO in the basal ganglia can be accounted for during image analysis

because it is a segregated brain area where tau accumulation is not expected. However, off-target binding in the choroid plexus can be problematic because of its close proximity to the hippocampal regions and entorhinal cortex. These are areas where tau accumulation typically begins in AD,33 meaning partial volume effects from off-target tracer accumulation in choroid plexus can complicate detection of early tau accumulation in hippocampus and entorhinal cortex.34,

35

Moreover, for any studies conducted without an MRI scan with which to co-register the [18F]AV1451 PET scan, we expect it would be challenging to identify precise structures such as entorhinal cortex and choroid plexus, and therefore difficult to distinguish true tau signal from off-target binding. There are also considerations about how off-target binding affects quantitative analysis of [18F]AV-1451 PET scans. The cerebellum is thought to be spared from tau pathology36 and our IHC results support this assertion (see Results section and Supporting Information). Reflecting this, cerebellum has been used as a reference region for analysis of [18F]AV-1451 PET data.30 However, since MAO is found in the cerebellum,37 Fowler found age-related increases in cerebellum MAO-B levels in her early MAO PET studies,38, 39 and MAOIs are being investigated for treatment of dementias such as AD,37 off-target binding of [18F]AV-1451 to MAO in the cerebellum could complicate its use as a reference region for this tracer. For all of these reasons, we were motivated to better understand the nature of [18F]AV-1451 binding to MAO.


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Off-target binding to MAO is perhaps not surprising given the structural similarities between [18F]AV-1451 (1) and a number of common MAO inhibitors containing a carboline core (Figure 5), and using our previously developed MAO activity assay40, 41 we determined that AV-1451 is indeed a non-selective inhibitor of MAO-A and –B. Though not a strong inhibitor (MAO-A: Ki = 25.00 ± 1.19 μM, MAO-B: Ki = 28.37 ± 1.22 μM), it is similar to structurally-related norharmane (2), an effective MAO inhibitor despite a relatively weak inhibitory constant (literature values: MAO-A: Ki = 2.2 µM; MAO-B Ki= 0.7-1.1 µM;42 our assay (see Supporting Information): MAOA: Ki = 19.63 µM; MAO-B Ki= 8.52 µM). We next investigated whether this association with MAO was contributing to the off-target [18F]AV-1451 PET signal using postmortem brain tissue sections from dementia subjects (2 x AD and 2 x DLB) as well as normal controls. We selected the substantia nigra because of the known off-target binding in this brain region, as well as cerebellum sections because it has been used as the reference region when analyzing [18F]AV-1451 PET data.30 Specific binding was identified using excess AV-1451 (500 nM) and found to be moderate in the dementia tissue samples (~50% in SN; ~10% in CBL). The residual signal is likely due to non-displaceable distribution of [18F]AV1451 in the tissue. Tissue sections for both SN and CBL from cognitively normal subjects did not show statistically significant differences between total and nonspecific [18F]AV-1451 binding, consistent with the expected lack of disease pathology in these subjects (tau or elevated MAO). Specific binding was ~400 times higher in diseased (AD and DLB) SN sections when compared to control sections; this increase was consistent, but to a lesser extent, for the CBL sections (Figure 2 and Supporting Information). We next co-incubated adjacent brain tissue samples with [18F]AV1451 and both high (500 µM) and low (500 nM) concentrations of deprenyl, an irreversible MAOB inhibitor. Deprenyl significantly lowered radioligand binding in diseased SN and CBL tissue sections by ~40 – 60% at both concentrations (Figure 2). In the CBL sections, this corresponded to larger displacement than could be attributed to specific binding alone. This suggests that deprenyl and AV-1451 are not competing for the same binding site on MAO, and possible involvement of a secondary binding site. There was no evidence of a dose-response relationship between [18F]AV-1451 binding and the two deprenyl concentrations tested (500 nM and 500 µM). For example, there was no significant difference between [18F]AV-1451 binding in the presence of low and high concentrations of deprenyl in the cerebellum. Interestingly, the low concentration



of deprenyl displaced more AV-1451 in substantia nigra than the high concentration, and we do not have an explanation for this finding. The two concentrations were chosen to span a range of doses and reveal any interactions that could be present. It is possible that the higher concentration of deprenyl (500 µM) may simply have been too high for this type of experiment, although we did not observe any deprenyl precipitation. Tissue sections for both SN and CBL from cognitively normal subjects did not have statistically significant differences in [18F]AV-1451 total binding compared to the MAO-B inhibition challenges (see Supporting Information). These results are consistent with an increase in MAO-B expression in disease, but low MAO-B expression in the normal brain.43 Immunoreactivity studies were next conducted and confirmed a significant increase in MAO-B abundance in diseased SN samples compared to controls (Figure 3), and a significant correlation was found between total [18F]AV-1451 binding and MAO-B immunoreactivity in this tissue (see Supporting Information). Despite the significant difference in MAO-B immunoreactivity in the diseased CBL compared to cognitively normal CBL, a similar correlation was not found with total [18F]AV-1451 binding. It is possible the trend could not be observed because of the high variability observed across individuals and/or the lack of tau NFTs in this region. Nevertheless, the high abundance of MAO-B in dementia CBL samples measured by immunoreactivity, in conjunction with the reduction of [18F]AV-1451 binding by deprenyl in autoradiography, strongly suggests specific binding to MAO-B in the cerebellum.


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A

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Figure 4. Averaged last four frames of dynamic PET scan in nonhuman primate brain (n=2 per study) in SUV (A) and as SUVR normalized to cerebellum (B).



Figure 5. [18F]AV-1451 and MAO inhibitors containing a carboline core. To further investigate the contribution of MAO binding to [18F]AV-1451 signals in the SN and CBL, PET scans were done in healthy nonhuman primates. Similar to human control subjects, [18F]AV-1451 distributes nonspecifically in the brain of the healthy rhesus macaque, and SUV is highest in the basal ganglia and cerebellum (see Supporting Information). Blocking with the reversible MAO-A inhibitor moclobemide resulted in a modest increase in SUVpeak in the cerebellum and basal ganglia, while pretreatment with the reversible MAO-B inhibitor lazabemide produced minor increases in the SUVpeak of the two regions. These increases are potentially due to blocking of [18F]AV-1451 binding sites in the periphery. Both inhibitors were found to ultimately reduce SUV in all brain regions at late time points as the radiotracer approached equilibrium (Figure 4A). Standardizing [18F]AV-1451 retention at the late time points to cerebellar uptake was found to disproportionately mask this decrease in SUV resulting from MAOI pretreatment in areas


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of known MAO expression the nonhuman primate brain (Figure 4B).44 These results are in contrast to the lack of differences in [18F]AV-1451 uptake between patients on MAO inhibitors and those not on medication (obtained using both SUV and SUVR) reported by Smith28 and Hansen27 (see Introduction). These discrepancies could be associated with species variations or the use of different MAO inhibitors; Smith also noted that their conclusions were preliminary because the number of patients on rasagiline was low, and data was acquired in a cross-sectional manner. In either case, it has been suggested that clinical interpretation of [18F]AV-1451 PET should not rely on use of the cerebellum as a reference region because of concerns regarding off-target binding.45 Our results support this suggestion and demonstrate that [18F]AV-1451 binding in the CBL is altered in vitro and in vivo by MAO inhibitors, and that there is an effect on data normalized to CBL reference region. These preclinical studies suggest that the blocking effects of MAO inhibitors being used to treat AD37 could reasonably translate to signal displacement in clinical imaging. The experiments described herein have three important limitations. First, enzymatic activity assays and tissue binding assays can be heavily influenced by experimental procedure. We aimed to provide a logical experimental design, in particular with our handling of post-mortem human tissue. For example, we avoided the use of ethanol in the autoradiography experiments, because it is strongly solubilizing and known to impact AV-1451 binding in such experiments.11,

13, 16

Secondly, the nonhuman primate PET scans did not include arterial input correction because of practical and regulatory considerations at our institution, and we have tried to remedy this by considering the later frames of the scan. Thirdly, the nonhuman primates used in these studies are healthy and thus have low levels of MAO-B. Future work could investigate whether there is a larger variation in [18F]AV-1451 SUVR when MAOIs are administered to nonhuman primates expressing higher levels of MAO-B (e.g. by using E. coli lipopolysaccharide (LPS) to induce a primate model of inflammation46). Despite these limitations, our in vitro and in vivo data are in agreement and indicate that i) AV-1451 inhibits monoamine oxidase activity in vitro; ii) AV-1451 binds to MAO-B on post-mortem tissue sections; and iii) in vivo, MAO inhibitors change the uptake of AV-1451 in the CBL, which is a reference region for [18F]AV-1451 PET used in the clinic.



Lastly, our interest in MAO imaging extends beyond its role as an off-target binding partner for AV-1451. MAO-B is a marker of astrocytosis, with evidence for its co-expression with amyloid plaques dating back to the early 1980’s.47,

48

Changes of MAO-B activity itself have been

investigated with irreversible inhibitors, reversible inhibitors, and substrates in PET imaging.49 It is quite unfortunate that not only does AV-1451 have an off-target binding partner, but that it is a binding partner that has a role in neurodegeneration. The immunoreactivity studies described herein confirm that MAO-B expression is increased in diseased SN and CBL sections, meaning observed changes in AV-1451 uptake in those regions are likely not due to the presence of tau NFTs alone. 4. CONCLUSIONS In conclusion, this work demonstrates that AV-1451 is a weak, nonselective inhibitor of monoamine oxidase and that [18F]AV-1451 off-target binding is, at least partially, attributable to MAO. From our in vitro and in vivo findings, and given known changes in MAO-B activity with age and neurodegeneration, we assert that prospective studies into the influence of MAO-B and different MAOIs on [18F]AV-1451 PET scans, are necessary. Moreover, given the presence of specific binding in the cerebellar reference region, in order to truly interpret SUVR, the community needs to have a more thorough understanding of the nature of [18F]AV-1451 binding in the cerebellum. 5. METHODS [18F]AV-1451 was produced as previously described.50 Details of all other experimental procedures as well as associated analytical data can be found in the Supporting Information. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.XXX. Experimental details for MAO inhibition assay, radiochemistry, autoradiography and immunohistochemistry tissue studies, nonhuman primate and human PET imaging protocols.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Allen F. Brooks: 0000-0003-3773-3024 Lindsey R. Drake: 0000-0001-8594-8922 Michael R. Kilbourn: 0000-0003-4100-4676 Peter J. H. Scott: 0000-0002-6505-0450 Author Contributions † L.R.D. and J.M.P.: Equal contribution. All authors contributed to writing this article. Notes The authors declare no competing financial interest. Funding Financial support for this work from the NIH (NIGMS: Pharmacological Sciences Training Program T32GM07767 (L.R.D., P.J.H.S)) and MICH-R Pilot Grant UL1TR002240 (A.F.B.) is gratefully acknowledged.

Acknowledgments We thank Phillip Sherman, Janna Arteaga, and Jenelle Stauff for conducting pre-clinical PET imaging. Abbreviations Used AD, Alzheimer’s disease; BG, basal ganglia; CBL, cerebellum; ChP, choroid plexus; DLB, dementia with Lewy bodies; EC, entorhinal cortex; MAO, monoamine oxidase; MAOI, monoamine oxidase inhibitor; MCI, mild cognitive impairment; MRI, magnetic resonance imaging; NFTs, neurofibrillary tangles; OC, occipital cortex; PET, positron emission tomography; SN, substantia nigra; TL, lateral temporal lobe.



References 1. Morbelli, S., Bauckneht, M., and Scheltens, P. (2017) Imaging Biomarkers in Alzheimer’s Disease: Added Value in the Clinical Setting, Q. J. Nucl. Med. Mol. Imaging 61, 360-371. 2. Henriques, A. D., Benedet, A. L., Camargos, E. F., Rosa-Neto, P., and Nóbrega, O. T. (2018) Fluid and Imaging Biomarkers for Alzheimer's Disease: Where we Stand and Where to Head to, Exp. Gerontol. 107, 169-177. 3. Giacobini, E., and Gold, G. (2013) Alzheimer disease therapy—moving from amyloid-β to tau, Nat. Rev. Neurol. 9, 677. 4. Lowe, V. J., Wiste, H. J., Senjem, M. L., Weigand, S. D., Therneau, T. M., Boeve, B. F., Josephs, K. A., Fang, P., Pandey, M. K., Murray, M. E., Kantarci, K., Jones, D. T., Vemuri, P., Graff-Radford, J., Schwarz, C. G., Machulda, M. M., Mielke, M. M., Roberts, R. O., Knopman, D. S., Petersen, R. C., and Jack Jr, C. R. (2018) Widespread Brain Tau and its Association with Ageing, Braak Stage and Alzheimer's Dementia., Brain 141, 271-287. 5. Villemagne, V. L., Fodero-Tavoletti, M. T., Masters, C. L., and Rowe, C. C. (2015) Tau imaging: early progress and future directions., Lancet Neurol. 14, 114-124. 6. Kolb, H. C., and Andrés, J. I. (2016) Tau positron emission tomography imaging, Cold Spring Harb. Perspect. Biol. 9, a023721. 7. Saint-Aubert, L., Lemoine, L., Chiotis, K., Leuzy, A., Rodriguez-Vieitez, E., and Nordberg, A. (2017) Tau PET imaging: present and future directions, Mol. Neurodegen. 12, 19. 8. Okamura, N., Harada, R., Ishiki, A., Kikuchi, A., Nakamura, T., and Kudo, Y. (2018) The Development and Validation of Tau PET Tracers: Current Status and Future Directions, Clin. Transl. Imaging 6, 305-316. 9. Villemagne, V. L., Doré, V., Burnham, S. C., Masters, C. L., and Rowe, C. C. (2018) Imaging tau and amyloid-β proteinopathies in Alzheimer disease and other conditions., Nat. Rev. Neurol. 14, 225-236. 10. Xia, C.-F., Arteaga, J., Chen, G., Gangadharmath, U., Gomez, L. F., Kasi, D., Lam, C., Liang, Q., Liu, C., Mocharla, V. P., Mu, F., Sinha, A., Su, H., Szardenings, A. K., Walsh, J. C., Wang, E., Yu, C., Zhang, W., Zhao, T., and Kolb, H. C. (2013) [18F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease, Alzheimers Dement 9, 666-676. 11. Marquié, M., Normandin, M. D., Vanderburg, C. R., Costantino, I. M., Bien, E. A., Rycyna, L. G., Klunk, W. E., Mathis, C. A., Ikonomovic, M. D., Debnath, M. L., Vasdev, N., Dickerson, B. C., Gomperts, S. N., Growdon, J. H., Johnson, K. A., Frosch, M. P., Hyman, B. T., and GómezIsla, T. (2015) Validating Novel Tau Positron Emission Tomography Tracer [F-18]-AV-1451 (T807) on Postmortem Brain Tissue., Ann. Neurol. 78, 787-800. 12. Sander, K., Lashley, T., Gami, P., Gendron, T., Lythgoe, M. F., Rohrer, J. D., Schott, J. M., Revesz, T., Fox, N. C., and Årstad, E. (2016) Characterization of Tau Positron Emission Tomography Tracer [18F]AV-1451 Binding to Postmortem Tissue in Alzheimer's Disease, Primary Tauopathies, and Other Dementias., Alzheimer's Dement. 12, 1116-1124. 13. Leuzy, A., Chiotis, C., Lemoine, L., Gillberg, P.-G., Almkvist, O., Rodriguez-Vieitez, E., and Nordberg, A. (2019) Tau PET Imaging in Neurodegenerative Tauopathies—Still a Challenge, Mol. Psychiatry, https://doi.org/10.1038/s41380-41018-40342-41388. 14. Klunk, W. E. (2018) Molecular imaging: What is right and what is an illusion?, Alzheimers Dement 10, 217-220. 15. Lemoine, L., Leuzy, A., Chiotis, K., Rodriguez-Vieitez, E., and Nordberg, A. (2018) Tau positron emission tomography imaging in tauopathies: The added hurdle of off-target binding, Alzheimer’s Dement 10, 232-236. 16. Marquie, M., Normandin, M. D., Meltzer, A. C., Chong, M. S. T., Andrea, N. V., AntonFernandez, A., Klunk, W. E., Mathis, C. A., Ikonomovic, M. D., Debnath, M., Bien, E. A., Vanderburg, C. R., Costantino, I., Makaretz, S., DeVos, S. L., Oakley, D. H., Gomperts, S. N., Growdon, J. H., Domoto-Reilly, K., Lucente, D., Dickerson, B. C., Frosch, M. P., Hyman, B. T.,


Page 16 of 19

Page 17 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Johnson, K. A., and Gomez-Isla, T. (2017) Pathological correlations of [F-18]-AV-1451 imaging in non-alzheimer tauopathies, Ann. Neurol. 81, 117-128. 17. Coakeley, S., Cho, S. S., Koshimori, Y., Rusjan, P., Ghadery, C., Kim, J., Lang, A. E., Houle, S., and Strafella, A. P. (2018) [18F]AV-1451 binding to neuromelanin in the substantia nigra in PD and PSP, Brain Struct. Funct. 223, 589-595. 18. Choi, J. Y., Cho, H., Ahn, S. J., Lee, J. H., Ryu, Y. H., Lee, M. S., and Lyoo, C. H. (2018) Off-Target 18F-AV-1451 Binding in the Basal Ganglia Correlates with Age-Related Iron Accumulation, J. Nucl. Med. 59, 117-120. 19. Hansen, A. K., Knudsen, K., Lillethorup, T. P., Parbo, P., Fedorova, T., Audrain, H., Bender, D., Borghammer, P., Landau, A. M., Ostergaard, K., and Brooks, D. J. (2016) In vivo imaging of neuromelanin in Parkinson's disease using 18F-AV-1451 PET, Brain 139, 2039-2049. 20. Marquié, M., Verwer, E. E., Meltzer, A. C., Kim, S. J. W., Agüero, C., Gonzalez, J., Makaretz, S. J., Chong, M. S. T., Ramanan, P., Amaral, A. C., Normandin, M. D., Vanderburg, C. R., Gomperts, S. N., Johnson, K. A., Frosch, M. P., and Gómez-Isla, T. (2017) Lessons learned about [F-18]-AV-1451 off- target binding from an autopsy-confirmed Parkinson’s case, Acta Neuropathol. Commun. 5, 75. 21. Lockhart, S. N., Ayakta, N., Winer, J. R., La Joie, R., Rabinovici, G. D., and Jagust, W. J. (2017) Elevated 18F-AV-1451 PET tracer uptake detected in incidental imaging findings, Neurology 88, 1095-1097. 22. Nagatsu, T., and Sawada, M. (2006) Molecular mechanism of the relation of monoamine oxidase B and its inhibitors to Parkinson's disease: possible implications of glial cells, J. Neural Transm., Suppl. 71, 53-65. 23. Proneth, B., and Conrad, M. (2018) Ferroptosis and necroinflammation, a yet poorly explored link, Cell Death Differ 26, 14-24. 24. Rombouts, F., Moechars, D., Andres, J. I., MacDonald, G., Chupakhin, V., Langlois, X., Bormans, G., and Declercq, L. (2016) [18F]JNJ-311, a novel tau PET ligand, pp MEDI-178, American Chemical Society. 25. Lemoine, L., Gillberg, P.-G., Nordberg, A., Svedberg, M., Stepanov, V., Jia, Z., Nag, S., Halldin, C., Huang, J., Tian, H., Ghetti, B., Okamura, N., Higuchi, M., and Nordberg, A. (2017) Comparative binding properties of the tau PET tracers THK5117, THK5351, PBB3, and T807 in postmortem Alzheimer brains, Alzheimers Res Ther 9, 96. 26. Saura, J., Kettler, R., Da Prada, M., and Richards, J. (1992) Quantitative enzyme radioautography with 3H-Ro 41-1049 and 3H-Ro 19- 6327 in vitro: localization and abundance of MAO-A and MAO-B in rat CNS, peripheral organs, and human brain, J. Neurosci. 12, 19771999. 27. Hansen, A. K., Brooks, D. J., and Borghammer, P. (2018) MAO-B Inhibitors Do Not Block In Vivo Flortaucipir ([18F]-AV-1451) Binding, Molecular Imaging and Biology 20, 356-360. 28. Smith, R., Schöll, M., Londos, E., Ohlsson, T., and Hansson, O. (2018) 18F-AV-1451 in Parkinson’s Disease With and Without Dementia and in Dementia with Lewy Bodies, Sci. Rep. 8, 4714. 29. Ng, K. P., Pascoal, T. A., Mathotaarachchi, S., Therriault, J., Kang, M. S., Shin, M., Guiot, M.-C., Guo, Q., Harada, R., Comley, R. A., Massarweh, G., Soucy, J.-P., Okamura, N., Gauthier, S., and Rosa-Neto, P. (2017) Monoamine oxidase B inhibitor, selegiline, reduces 18FTHK5351 uptake in the human brain, Alzheimers Res Ther 9, 25. 30. Maass, A., Landau, S., Baker, S. L., Horng, A., Lockhart, S. N., La Joie, R., Rabinovici, G. D., and Jagust, W. J. (2017) Comparison of Multiple tau-PET Measures as Biomarkers in Aging and Alzheimer's Disease, Neuroimage 157, 448-463. 31. Vermeiren, C., Motte, P., Viot, D., Mairet-Coello, G., Courade, J.-P., Citron, M., Mercier, J., Hannestad, J., and Gillard, M. (2018) The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases, Mov. Disord. 33, 273-281.



32. Drake, L. R., Pham, J., Brooks, A., Koeppe, R., and Scott, P. (2019) MAO-B Inhibitor Deprenyl Displaces [18F]AV1451 binding on human brain tissue sections and alters uptake in Nonhuman Primate Brain, J. Nucl. Med. 60 (Suppl 1), 487. 33. Hu, W., Wu, F., Zhang, Y., Gong, C., Iqbal, K., and Liu, F. (2017) Expression of Tau Pathology-Related Proteins in Different Brain Regions: A Molecular Basis of Tau Pathogenesis., Front Aging Neurosci. 9, 311. 34. Baker, S., Lockhart, S., Maass A, and Jagust, W. (2017) Partial volume effects and medial temporal lobe tau quantitation with PET, Alzheimer's Dement. 13 (Suppl), P4-052. 35. Baker, S. L., A, M., and Jagust, W. J. (2017) Considerations and code for partial volume correcting [18F]-AV-1451 tau PET data, Data Brief 15, 648-657. 36. Larner, A. (1997) The cerebellum in Alzheimer's disease., Dement. Geriatr. Cogn. Disord. 8, 203-209. 37. Cai, Z. (2014) Monoamine oxidase inhibitors: Promising therapeutic agents for Alzheimer's disease (Review), Mol. Med. Rep. 9, 1533-1541. 38. Fowler, J. S., MacGregor, R. R., Wolf, A. P., Arnett, C. D., Dewey, S. L., Schlyer, D., Christman, D., Logan, J., Smith, M., Sachs, H., Aquilonius, S. M., Bjurling, P., Halldin, C., Hartvig, P., Leenders, K. L., Lundqvist, H., Oreland, L., Stålnacke, C.-G., and Långström, B. (1987) Mapping Human Brain Monoamine Oxidase A and B with 11C-Labeled Suicide Inactivators and PET, Science 235, 481-485. 39. Fowler, J. S., Volkow, N. D., Wang, G.-J., Logan, J., Pappas, N., Shea, C., and MacGregor, R. R. (1997) Age-related increases in brain monoamine oxidase B in living healthy human subjects, Neurobiol. Aging 18, 431-435. 40. Drake LR, Brooks AF, Mufarreh AJ, Pham JM, Koeppe RA, Shao X, Scott PJH, and MR., K. (2018) Deuterium Kinetic Isotope Effect Studies of a Potential in Vivo Metabolic Trapping Agent for Monoamine Oxidase B., ACS Med. Chem. Lett. 9, 3024-3027. 41. Brooks AF, Drake LR, Shao X, Zhao A, Scott PJH, and MR., K. (2018) Evaluation of Enzyme Substrate Radiotracers as PET/MRS Hybrid Imaging Agents., ACS Med. Chem. Lett. 9, 1140-1143. 42. van Amsterdam J, Talhout R, Vleeming W, and A., O. (2006) Contribution of Monoamine Oxidase (MAO) Inhibition to Tobacco and Alcohol Addiction., Life Sci., 1969-1973. 43. Schedin-Weiss, S., Inoue, M., Hromadkova, L., Teranishi, Y., Yamamoto, N. G., Wiehager, B., Bogdanovic, N., Winblad, B., Sandebring-Matton, A., Frykman, S., and Tjernberg, L. O. (2017) Monoamine Oxidase B is Elevated in Alzheimer Disease Neurons, is Associated with γSecretase and Regulates Neuronal Amyloid β-peptide Levels, Alzheimers Res. Ther. 9, 57. 44. Brooks, A. F., Shao, X., Quesada, C. A., Sherman, P., Scott, P. J. H., and Kilbourn, M. R. (2015) In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase‐A and -B Enzymatic Activity, ACS Chem. Neurosci. 6, 1965-1971. 45. Chen, J., Pirraglia, E., Okamura, N., Rusinek, H., and de Leon, M. J. Alzheimer’s Disease Neuroimaging Initiative. (2018) Quantitative Evaluation of Tau PET Tracers 18F-THK5351 and 18F-AV-1451 in Alzheimer’s Disease with Standardized Uptake Value Peak-alignment (SUVP) Normalization, Eur. J. Nucl. Med. Mol. Imaging 45, 1596-1604. 46. Hannestad, J., Gallezot J-D., Schafbauer, T., Lim, K., Kloczynski, T., Morris, E. D., Carson, R. E., Ding, Y.-S., and Cosgrove, K. P. (2012) Endotoxin-induced systemic inflammation activates microglia: [11C]PBR28 positron emission tomography in nonhuman primates, Neuroimage 63, 232-239. 47. Adolfsson, R., Gottfries, C.-G., Oreland, L., Wiberg, Å., and Winblad, B. (1980) Increased Activity of Brain and Platelet Monoamine Oxidase in Dementia of Alzheimer Yype, Life Sci. 27, 1029-1034. 48. Saura J, Luque JM, Cesura AM, Da Prada M, Chan-Palay V, Huber G, Löffler J, and JG., R. (1994) Increased Monoamine Oxidase B Activity in Plaque-associated Astrocytes of Alzheimer Brains Revealed by Quantitative Enzyme Radioautography., Neuroscience 62, 15-30.


Page 18 of 19

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49. Narayanaswami, V., Drake, L. R., Brooks, A. F., Meyer, J. H., Houle, S., Kilbourn, M. R., Scott, P. J. H., and Vasdev, N. (2019) Classics in Neuroimaging: Development of PET Tracers for Imaging Monoamine Oxidases, ACS Chem. Neurosci. 10, 1867-1871. 50. Mossine, A. V., Brooks, A. F., Henderson, B. D., Hockley, B. G., Frey, K. A., and Scott, P. J. H. (2017) An updated radiosynthesis of [18F]AV1451 for tau PET imaging, EJNMMI Radiopharm Chem 2, 7.

Table of Contents Graphic

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18F

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PET signal due to both tau and off-target binding


[ 18F]AV-1451

Identification of AV-1451 as a Weak, Nonselective Inhibitor of

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