Monoamine oxidase binding not expected to significantly affect [18F]flortaucipir PET interpretation

Purpose [18F]-labeled positron emission tomography (PET) radioligands permit in vivo assessment of Alzheimer’s disease biomarkers, including aggregated neurofibrillary tau (NFT) with [18F]flortaucipir. Due to structural similarities of flortaucipir with some monoamine oxidase A (MAO-A) inhibitors, this study aimed to evaluate flortaucipir binding to MAO-A and MAO-B and any potential impact on PET interpretation. Methods [18F]Flortaucipir autoradiography was performed on frozen human brain tissue slices, and PET imaging was conducted in rats. Dissociation constants were determined by saturation binding, association and dissociation rates were measured by kinetic binding experiments, and IC50 values were determined by competition binding. Results Under stringent wash conditions, specific [18F]flortaucipir binding was observed on tau NFT-rich Alzheimer’s disease tissue and not control tissue. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A; pre-treatment with MAO inhibitor pargyline did not impact uptake or wash-out of [18F]flortaucipir. [18F]Flortaucipir bound with low nanomolar affinity to human MAO-A in a microsomal preparation in vitro but with a fast dissociation rate relative to MAO-A ligand fluoroethyl-harmol, consistent with no observed in vivo binding in rats of [18F]flortaucipir to MAO-A. Direct binding of flortaucipir to human MAO-B was not detected in a microsomal preparation. A high concentration of flortaucipir (IC50 of 1.3 μM) was found to block binding of the MAO-B ligand safinamide to MAO-B on microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro. Conclusion These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [18F]flortaucipir. Supplementary Information The online version contains supplementary material available at 10.1007/s00259-022-05822-9.

[ 18 F]Flortaucipir is structurally similar to known reversible inhibitors of monoamine oxidase A (MAO-A), including harmine and fluoroethyl-harmol. Off-target binding has been noted in areas of the brain where aggregated tau is unexpected, and there have been conflicting reports in the literature about possible MAO-A and MAO-B binding relationships with [ 18 F]flortaucipir [18][19][20]. Furthermore, PET signal for the putative tau tracer [ 18 F]THK5351 has been reported to be greatly reduced following treatment with the MAO-B inhibitor selegiline (deprenyl) [21]. The present study aimed to investigate and characterize any binding relationship between [ 18 F]flortaucipir and MAO-A or MAO-B in vivo using PET imaging and by in vitro competition and saturation binding experiments. [ 18 F]Flortaucipir was produced as described previously [22]. [ 18 F]Fluoroethyl-harmol was produced following the procedure as described by Schieferstein et al. [23].

[ 18 F]Flortaucipir autoradiography: comparison of stringent and mild wash conditions
Autoradiography was performed on frozen human brain slices from AD donors (N = 3, PHF tau-rich as determined by AT8 immunohistochemistry) and age-matched non-AD control donors (N = 6, devoid of tau NFTs and beta-amyloid neuritic plaques as determined by immunohistochemistry). Brains slices were obtained from the National Disease Research Interchange (Philadelphia, PA).

[ 18 F]Flortaucipir autoradiography: competition binding assay with MAO-A and MAO-B inhibitors
Autoradiography competition binding assay of [ 18 F]flortaucipir binding to normal human brain was used to determine the IC 50 values of flortaucipir, MAO-A inhibitors clorgyline (Sigma-Aldrich) and fluoroethyl-harmol (prepared by alkylation of harmol [Alfa Aesar] with 1-bromo-2-fluoroethane as described by Ng et al. [21]), and MAO-B inhibitors deprenyl (Sigma-Aldrich) and safinamide (TCI). Adjacent frozen sections (10 μm thick) from a normal human temporal cortex (sourced from National Disease Research Interchange) were incubated with a fixed concentration of [ 18 F]flortaucipir (0.74MBq [20 μCi] in 500 μL, 1.4 nM each slide) with varying concentrations of competing compound (log serial dilution from 10 μM to 0.001 μM) in 2.5% DMSO/2.5% EtOH/PBS, pH 7.4. After 60-min incubation at room temperature, PBS washes (4 × 2 min) were used to remove any unbound ligand. After drying under ambient conditions, the sections were placed in a cassette and exposed to a phosphor imaging plate overnight. The plate was then scanned with a GE Typhoon FLA7000 Bio-Imaging System. Signal intensity (counts/pixel) on each section was measured using Multi Gauge V3.0 Imaging software. Total binding was evaluated by nonlinear regression using GraphPad Prism v8 to determine IC 50 values.

In vivo PET imaging
All procedures in this study followed the guidelines of the Institutional Animal Care and Use Committee of the University of the Sciences, Philadelphia, PA. A Siemens Inveon Multimodality Scanner was used for micro-PET/computed tomography (CT) imaging. Male wild-type Sprague-Dawley rats (128 ± 58 g) were anesthetized with 3% isoflurane with oxygen. Rats were administered [ 18 F]fluoroethyl-harmol or [ 18 F] flortaucipir via tail vein injection  in a total volume of 200 μL). For blocking studies, rats were pre-treated with pargyline (Sigma-Aldrich, 50 mg/kg i.p.) 30 min prior to scans; control rats were pre-treated with 0.9% saline injection (Hospira, i.p.). A short high-resolution CT scan was first conducted for anatomical registration, followed by a 120-min dynamic PET scan. PET images were generated for each minute of the acquisition time. Uptake of the tracers was determined by visually drawing regions of interest based on the fused PET/CT images, and corresponding activity values were determined using Inveon Research Workplace software. All values are represented as % injected dose per gram (ID/g). A total of 6 rats were studied (3 control, 3 pre-treated) for [ 18

PHF preparation
Purified, soluble PHF was isolated from AD brain tissue using a protocol modified from the procedure described by Jicha et al. [24]. Briefly, homogenized AD cortex underwent high pressure batch-gas expansion using a Parr Cell disruption bomb and centrifuged at 28 kg. Soluble PHF was isolated from the supernatant by affinity chromatography using an MC1-Affigel 10 column (25 mL flow rate), with a high guard column (4 cm) of Sepharose 400 Superflow, recycling supernatant twice through the column over 18-20 h at 4 °C. Bound PHF was eluted with two column volumes of 3M potassium thiocyanate. Isolated PHF was analyzed in a tau MC-1 ELISA and a tau AT8 ELISA, which recognizes tau phosphorylated at both Ser 202 and Thr 205 [3].

K d and IC 50 determination
The dissociation constant (K d ) was determined by saturation binding, in which the total and non-specific binding of the radioligand was measured at various concentrations. Human recombinant MAO-A, MAO-B, and control microsomes were acquired from Sigma (M7316, M7441, and M7566, respectively). All microsome preparations were at a concentration of 5 mg protein/mL. The reaction mixture (250 μL) contained protein target and 18 F-radioligand, serially diluted in PBS, pH 7.4; assays were performed in PBS (0.01% bovine serum albumin [BSA]) in 96-well polypropylene microplates. Non-specific binding (NSB) was defined as radioligand binding in the absence of target or in the presence of an excess of non-radioactive compound selective for the target: T808 (10 μM, synthesized at Avid Radiopharmaceuticals according to published procedures) [25] for PHF, clorgyline (1-10 μM) for MAO-A, and deprenyl (1-10 μM) for MAO-B. After incubation (1.5 h at 37 °C), the bound radioactivity was harvested onto Millipore MultiScreen® HTS FB filter plates by vacuum filtration (Millipore MultiScreen® HTS Vacuum Manifold), followed by 5 PBS washes. Filters were assayed for radioactivity in a Wizard 2480 automatic gamma-counter (Perkin Elmer). Data were analyzed by nonlinear regression analysis (GraphPad Prism) to determine K d for the radioligand.
For non-radioactive compounds, K d was determined by saturation binding in 96-well polypropylene microplates; the reaction mixture (250 μL) contained a fixed amount of protein and 12 concentrations of ligand and PBS (0.02% BSA). After incubation (1.5 h at 37 °C), bound ligand was harvested by vacuum filtration onto a glass fiber filter plate (Pall® Acroprep™ Advance 96-well 1.0 μm), using a Millipore MultiScreen® HTS Vacuum Manifold, followed by 5 PBS washes. Bound ligand was then eluted into a 96-deepwell 700 μL polypropylene plate with 250 μL internal standard in methanol by centrifugation at 4000 RPM for 5 min.
Each sample (15 μL) was analyzed via liquid chromatography-mass spectrometry (LC-MS, see supplementary information for more detail). Data were analyzed by nonlinear regression analysis (GraphPad Prism).
IC 50 values for each compound were determined by competition binding. The reaction mixture (250 μL) contained a set amount of protein and ligand as well as a decreasing amount of inhibitor, from 10 μM to 0.32 nM (1/2 log dilution series), in PBS containing 0.02% BSA in 96-well polypropylene microplates. After incubation for 1.5 h at 37 °C, bound ligand was harvested and analyzed by LC-MS as described above. IC 50 values were determined by nonlinear regression analysis using GraphPad Prism.

Kinetic binding study
The association (k on ) and dissociation (k off ) rates were determined by kinetic binding experiments in which binding of a radioligand to a target (PHF tau or MAO-A or MAO-B microsomal preparations) was measured at multiple timepoints. The reaction mixture (250 μL) contained protein target and 18 F-radioligand (1 nM); assays were performed in PBS containing 0.01% BSA in 96-well polypropylene microplates. At each timepoint of the 37 °C incubation, the bound radioactivity was harvested by vacuum filtration onto Millipore MultiScreen® HTS FB filter plates, using a Millipore MultiScreen® HTS Vacuum Manifold, followed by 5 PBS washes. Once the equilibrium signal was reached, dissociation was initiated by adding excess non-radioactive compound. Measurements continued until no binding was detected. Filters containing bound [ 18 F]flortaucipir were assayed and analyzed for radioactivity as previously noted.

Results
[ 18 F]Flortaucipir autoradiography was performed on frontal cortex and striatum frozen human brain tissue slices from multiple AD and control donors to assess tau NFT and off-target binding (Fig. 1). Under stringent washing conditions (30% and 70% EtOH), [ 18 F]flortaucipir binding was observed only on tau NFT-rich AD tissue, not on control tissue. Autoradiography signal on AD tissue was found to be saturable in the presence of non-radioactive flortaucipir. However, when mild washing conditions were used, binding of [ 18 F]flortaucipir was observed on both AD and control tissues, in an off-target pattern different than that observed using stringent washing conditions, and for which binding was only partially saturable by 1 μM flortaucipir.
To further understand the off-target binding observed under mild wash conditions, inhibition of [ 18 F]flortaucipir binding by MAO-A or MAO-B inhibitors was evaluated on temporal cortex brain sections from control subjects, devoid of tau NFTs, and beta-amyloid neuritic plaques ( Fig. 2A PET studies were conducted in rats to compare retention of a selective and reversible MAO-A tracer, [ 18 F]fluoroethylharmol, to that of [ 18 F]flortaucipir in both control and 50 mg/kg pargyline (an irreversible MAO-A/B inhibitor) pretreated rats (Fig. 3A, 3B). In control rats, [ 18 F]fluoroethylharmol crossed the blood-brain barrier, reached a peak of 2.3 %ID/g at 2 min, and was retained in the brain throughout the scan period with 2.1 %ID/g still present at 110 min, consistent with its strong binding relationship with MAO-A. However, for [ 18 F]fluoroethyl-harmol in the pargyline pretreated group, peak brain activity reached 2.4 %ID/g at 2 min, followed by a rapid washout to 1.0 %ID/g by 20 min. In contrast, [ 18 F]flortaucipir displayed nearly identical brain uptake with rapid clearance in both the control and pargyline groups ( Studies using the putative tau tracer [ 18 F]THK5351 have shown that its affinity toward MAO-B confounds its quantification of tau by PET [21]. To explore the potential affinity of flortaucipir for MAO-B, in vitro saturation binding of [ 18 F] flortaucipir and [ 18 F]THK5351 to MAO-B microsomes was conducted (Fig. 4)  Additional saturation binding experiments were conducted at higher flortaucipir concentrations in a liquid chromatography-mass spectrometry (LC-MS) direct binding assay (Fig. 5). Again, the K d for flortaucipir binding to recombinant MAO-B microsomes could not be determined; no specific binding was observed as evidenced by the lack of separation between total binding and non-specific binding curves. By comparison, the K d for the binding of safinamide, a selective and reversible MAO-B ligand, to recombinant MAO-B was 57 nM with a B max of 446 pmol ligand/mg total protein when binding to control microsome (absence of target) is used to define NSB.

Discussion
This study aimed to characterize any binding relationship of [ 18   The affinity and specificity of flortaucipir have been well characterized with [ 18 F]flortaucipir PET images in AD patients showing a pattern of distribution consistent with the pathological observations of Braak [26] and others for the distribution of tau NFTs in AD brains [14][15][16][17]. However, localization of [ 18 F]flortaucipir is seen also in striatum and choroid plexus in both AD patients and those with no evidence of disease [27]. It is possible that some of the signal in the choroid plexus can spill over into adjacent regions [27,28]. The source of this choroid plexus binding is not known but may reflect binding to iron/calcification or, less likely, Biondi bodies [29,30]. Presumed off-target elevations of PET signal for multiple tau PET tracers, including [ 18 F]flortaucipir, [ 18 F]MK-6240, and [ 18 F]THK5351, have also been observed in structures rich in neuromelanin and melanin-containing cells [29,[31][32][33][34]. These regions, however, reside outside of the AD-associated neocortical areas examined for visual interpretation, and thus, the observed  [31,35,36]. However, none of the aforementioned potential off-target binding sites have the same potential to be confused with tau deposition in AD as would binding to MAO-B in activated astrocytes.
Several other tau tracers in development have exhibited off-target binding presumably due to interaction with MAO-B [21,37,38]. This has raised the question whether [ 18 F]flortaucipir also interacts with MAO-B; thus far, conflicting evidence has failed to clarify any possible interaction [20,[38][39][40]. [ 18 F]THK5351, a radiotracer that has been investigated as a possible tau imaging agent, is known to bind to both NFT tau and MAO-B [41,42], and MAO-B inhibitors rasagiline and selegiline (deprenyl) significantly attenuate the uptake of [ 18 F]THK5351 in humans [21,43]. MAO-B interaction could impact the interpretation of PET scans for tau-targeted radioligands due to signal overlap, as MAO-B is expressed by reactive astrocytes that often colocalize with tau NFTs [44][45][46][47][48].
Concerns about off-target binding associations to MAO-B or MAO-A have prompted [ 18 F]flortaucipir PET studies in non-human primates to elucidate potential binding to MAOs [18,49]. We do not find the data in these studies to be consistent with specific in vivo binding and retention of flortaucipir to MAOs or specific displacement of flortaucipir binding by known MAO inhibitors in any brain region. Drake et al. [18] acknowledged that in their study, flortaucipir distributed non-specifically in the NHP brain; the washout curves for hippocampus, cerebellum, thalamus, basal ganglia, and cortex appear indistinguishable from each other (as shown in Drake et al. [18] Supplemental Information, Fig. S5). This pattern of distribution is in contrast to reported MAO-B ligands such as [ 11 C]deprenyl-d 2 [50] and [ 18 F]FSL25.1188 [51], which demonstrated high signal in both thalamus and basal ganglia, and reduced retention in the cerebellum or MAO-A ligands such as [ 11 C]harmine [52], which demonstrate higher activity in areas such as the thalamus and putamen relative to the cerebellum. In these three studies [50][51][52], activity in areas of high MAO expression such as thalamus and basal ganglia is significantly higher (1.5-to 2-fold) than in cerebellum, an area of low MAO expression [53], whereas a similar pattern is not observed in the study by Drake et al. In the non-human primate study by Hostetler et al. [49], a lack of regional differentiation for flortaucipir is also observed; however, in that study, evaluation of off-target MAO binding via blocking studies using specific inhibitors was not performed. A decreased distribution volume under self-blocking conditions is observed and is proposed to be evidence of off-target binding; however, the study does not identify possible causes, such as binding to MAOs. As a whole, these [ 18 F]flortaucipir PET studies probing potential off-target binding to MAOs have not convincingly demonstrated an association in vivo, prompting the current study.
In PET imaging studies using normal rodents, [ 18 F]flortaucipir binding to MAO-B could not be evaluated due to low levels of MAO-B expression. In autoradiography studies of normal human brain tissue using mild aqueous wash conditions, there was minimal displacement of [ 18   The results from our saturation binding experiments disagree with results published by Vermeiren et al. [20]. Although we also observe a 2.0 nM K d for [ 18 F]flortaucipir binding to recombinant human MAO-A, we found no evidence of significant binding to MAO-B. Vermeiren et al. used excess non-radioactive flortaucipir to define nonspecific binding [20], whereas we chose control microsomes devoid of recombinant MAO-B. We found that using excess flortaucipir to define NSB in filtration binding assays reduced [ 18 F]flortaucipir non-specific binding below that of the control microsomes and produced an artifactual specific binding curve in buffer only (without any MAO or PHF). Under similar conditions as Vermeiren et al. ( Supplemental  Information, Fig. S2), the observed signal represents background binding of [ 18 F]flortaucipir to the filter; this is then reduced by adding excess non-radioactive flortaucipir. Based on these results, we believe that the previously reported K d by Vermeiren et al. may also be artifactual.
Consistent with the absence of or low interaction with MAO-B in our studies, a retrospective analysis, Hansen et al. [19] found that MAO-B inhibitors at therapeutic concentrations did not significantly affect [ 18 F]flortaucipir binding in patients with Parkinson's disease, in which MAO-B inhibitors are used to treat the disease. This finding is further supported by a clinical trial reported by Matthews et al. [40], in which 24 weeks of treatment with MAO-B inhibitor, rasagiline, in patients with AD did not result in changes of flortaucipir binding in cortical regions. They did report modest uniform reductions in [ 18 F]flortaucipir signal in subcortical regions with high MAO-B expression, such as the striatum and nucleus accumbens [54]; however, there were no observed decreases in cortical regions with known MAO-B expression, suggesting that affinity of [ 18 F]flortaucipir for MAO-B is weak and importantly much weaker than its affinity for tau [40].
Additionally, studies have demonstrated [ 18 F]flortaucipir binding in the affected temporal tip in PET scans of patients with semantic variant primary progressive aphasia (svPPA), a disease characterized by TDP-43 deposition [35,55], raising the concern that [ 18 F]flortaucipir may be binding to TDP-43 or MAO-B associated with astrogliosis in these cases. However, Schaeverbeke et al. [35] found no evidence that [ 18 F] flortaucipir bound to TDP-43 in autopsy tissue from svPPA patients and no effect of blocking with the MAO-B inhibitor deprenyl on [ 18 F]flortaucipir binding by autoradiography [35]. Pascual et al. [55] also demonstrated that the [ 18 F]flortaucipir PET scan for an svPPA patient was unaffected by pre-treatment with deprenyl, an MAO-B inhibitor. These data from Pascual and Schaeverbeke are consistent with our preclinical data and clinical studies from Hansen [19] and Matthews [40] that [ 18 F]flortaucipir does not bind to MAO-B.
A possible interaction between [ 18 F]flortaucipir and MAO-A would be of much less concern with respect to imaging with [ 18 F]flortaucipir; unlike MAO-B, MAO-A is largely expressed in neurons, not astrocytes [56,57], and thus should not impact PET interpretation. Due to the structural similarity between [ 18 F]flortaucipir and the MAO-A ligands harmine and fluoroethyl-harmol, we also sought to characterize the binding of [ 18 F]flortaucipir to MAO-A. In autoradiography experiments, displacement of [ 18 F]flortaucipir binding by MAO-A ligands could be observed only using mild aqueous washing conditions and even then, only weakly (IC 50 0.1-1 μM). However, [ 18 F]flortaucipir bound to MAO-A accounted for ~50% of binding observed, while the remaining was non-specific and non-saturable since it could not be displaced by high levels of non-radioactive flortaucipir. In vivo PET experiments in rats revealed no evidence of [ 18 F]flortaucipir binding to MAO-A. In vitro studies revealed low nanomolar affinity of [ 18 F]flortaucipir for MAO-A in microsomes, but kinetic binding studies indicated that the off rate of [ 18 F]flortaucipir from MAO-A is much faster than [ 18 F]flortaucipir from PHF tau or [ 18 F]fluoroethyl-harmol from MAO-A. This fast dissociation rate from MAO-A may explain why binding of flortaucipir to MAO-A was not observed in PET imaging studies in animals, and consequently, it would not be expected that [ 18 F]flortaucipir would produce images of MAO-A in humans.
In summary, autoradiography experiments showed [ 18 F] flortaucipir localization or binding in tau-rich areas of AD tissue but not in normal control tissue under stringent wash conditions. In direct binding studies, flortaucipir had low nanomolar affinity toward MAO-A but with an off rate 9 times faster than for tau and PET imaging showed no significant retention consistent with binding to MAO-A in a rat model, in contrast to MAO-A ligand [ 18 F]fluoroethylharmol. Although we could not detect any direct binding of [ 18 F]flortaucipir to MAO-B or significant displacement from tissue by MAO-B ligands, a high concentration of flortaucipir blocked safinamide binding to MAO-B microsomes indicating that, at micromolar concentrations, flortaucipir may bind weakly to MAO-B in vitro. Overall, the in vitro and in vivo data presented here suggest that neither MAO-A nor MAO-B binding contribute significantly to [ 18 F]flortaucipir PET images. The present study, together with human PET blocking studies [40,55], clearly show that MAO binding cannot account for the flortaucipir PET signal. In conjunction with image to autopsy studies confirming the association and colocation of flortaucipir PET signal with brain NFTs, these data firmly support the interpretation of flortaucipir PET signal as an indicator of distribution and density of tau in the cortex of patients suspected of AD.