Autoradiography validation of novel tau PET tracer [F-18]-MK-6240 on human postmortem brain tissue

The recent development of several novel positron emission tomography (PET) tracers tailored to detect tau in the brain has opened the opportunity of using them to improve diagnostic accuracy in Alzheimer disease (AD) and related tauopathies, and to allow reliable quantification of brain tau burden and tracking of disease progression by in vivo neuroimaging [15, 35].

Emerging data from early studies -including our own- on postmortem material with the most validated thus far, [F-18]-AV-1451 (T807, Flortaucipir), have shown that this ligand binds with strong affinity to paired helical filament (PHF)-tau aggregates in AD brains and those that form as a function of age [20–22, 27, 35], closely matching the stereotypical spatiotemporal progression of neurofibrillary tangles (NFT) as described by Braak [3]. In agreement with these observations, patients clinically diagnosed with dementia of AD type and mild cognitive impairment (MCI) exhibit significantly higher in vivo [F-18]-AV-1451 retention than cognitively normal individuals in regions that are known to contain an elevated burden of tau lesions in AD [4, 6, 7, 11, 16, 25, 28, 33]. The overall utility of this tracer for in vivo selective and reliable detection of tau aggregates in non-AD tauopathies, however, seems very limited with the exception of certain tau mutations causing frontotemporal lobar degeneration (FTLD) characterized by tau aggregates [26] that contain all six isoforms of tau (three-repeat (3R) and four-repeat (4R)) [14] with PHF ultrastructure resembling NFT found in AD. We and others have shown that [F-18]-AV-1451 has low affinity for tau aggregates that contain primarily 4R tau with straight filament ultrastructure that predominate in tauopathies such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and most cases of FTLD. We also demonstrated the existence of robust [F-18]-AV-1451 off-target binding to melanin- and neuromelanin-containing cells and some weaker binding to blood components [21, 22]. Controversy exists as to whether AV-1451 may also exhibit significant nonspecific binding to MAO enzymes [12, 15, 17, 30], as it has been recently demonstrated for other tau PET tracers like THK-5351 [13, 24].

Several second-generation tau tracers have more recently been reported. The one that has garnered most attention and is largely considered to have most promise is [F-18]-MK-6240 from the Merck Translational Biomarkers team [15, 32]. To date, very limited information is available about the binding properties of this tracer. Merck’s researchers performed in vitro binding screens against a wide panel of known receptor, transporter, and enzyme targets but conducted only limited autoradiography studies on AD and control brain specimens [15]. Yet, this ligand is quickly making its way into observational studies and clinical trials. Thus, a comprehensive postmortem validation of MK-6240 is critical for determining its usefulness for antemortem diagnosis and staging of AD and other tauopathies, and to understand exactly what [F-18]-MK-6240 PET positivity means in terms of neuropathological substrate. Because MK-6240 was initially screened in vitro for NFT binding affinity using AD brain homogenates rich in NFT and an amyloid plaque tracer for counterscreening [15], and seemed to bind to the same site as AV-1451 in that tissue material, we predicted that, just like AV-1451, MK-6240 would exhibit high binding affinity and selectivity for PHF-tau lesions relative to non-PHF-tau lesions, Aβ deposits and α-synuclein and transactive response DNA binding protein-43 (TDP-43) aggregates.

To validate the site/s of MK-6240 binding and determine whether there is off-target binding, we examined the regional and substrate-selective autoradiograhic patterns of [F-18]-MK-6240 in postmortem brain, retina and skin tissue samples. Cases with a definite pathological diagnosis of AD, FTLD-tau (PiD, PSP, CBD), chronic traumatic encephalopathy (CTE), FTLD-TDP-43, dementia with Lewy bodies (DLB), cerebral amyloid angiopathy (CAA), metastatic melanoma, brain hemorrhages, and elderly controls free of neurodegenerative diseases were studied. Most of these cases had also been included in our previous validation studies of [F-18]-AV-1451 [21, 22], giving us the opportunity to directly compare the autoradiographic binding patterns of both tracers in comparable tissue samples.

Recently multiple novel PET tracers have been reported, tailored to allow detection of tau pathology in the human living brain. The status quo is largely to use PET ligands in patients carrying a particular clinical diagnosis and then simply accept, in a somewhat circular fashion, that what the scan shows is representative of the underlying disease process. However, the correct identification of biological targets of imaging agents is an essential requirement for considering them as disease-specific and progression-specific biomarkers. Validating the underlying neuropathological binding substrate/s and identifying potential off-target binding of these novel tau ligands is critical for the accurate interpretation of their in vivo PET imaging behavior. We have carefully characterized the autoradiographic binding patterns of novel tau tracer [F-18]-MK-6240 in a collection of postmortem tissue samples representing a broad spectrum of neurodegenerative disorders. Our observations derived from combined [F-18]-MK-6240 sensitive autoradiography and immunohistochemistry, using this compound at similar concentrations used in vivo for PET studies, indicate that MK-6240 has high binding affinity for tau aggregates in AD brain tissue, but does not seem to bind to a significant extent to neuronal and glial tau aggregates in non-AD tauopathies such as PiD, PSP, CBD or CTE, or to Aβ, α-synuclein or TDP-43-containing lesions. These findings strongly suggest that tau in tangles of AD has a unique conformation that is recognized by this tracer (in keeping with the selection process for development of this compound as a lead imaging agent). Strong binding of [F-18]-MK-6240 to neuromelanin- and melanin-containing cells and some weaker binding to brain hemorrhagic lesions was also identified, pointing to these substrates as off-target binding sites of MK-6240. Overall, [F-18]-MK-6240 autoradiographic binding patterns closely resembled those of tau PET tracer [F-18]-AV-1451. Importantly, [F-18]-MK-6240 and [F-18]-AV-1451 binding signals were only very weakly displaced using autoradiography competition with unlabeled selective MAO-B inhibitor deprenyl, suggesting that these two tracers have low binding affinity for MAO enzymes in the human brain.

MK-6240 was identified as a potential imaging agent by screening using cortical homogenates from AD tissue rich in NFT as the binding target and an amyloid plaque tracer for counter-screen [15]. Based on this, we anticipated that MK-6240 would preferentially bind to tau lesions in the form of NFT in AD over other tau aggregates in non-AD tauopathies or lesions primarily made of Aβ, α-synuclein or TDP-43. To date, only very limited data from human in vivo [F-18]-MK-6240 PET imaging studies have been published [2, 19]. Results from these early studies point to a promising 2- to 3-fold higher in vivo [F-18]-MK-6240 retention in neocortical and medial temporal brain regions of AD patients compared to elderly cognitively normal individuals [19]. Because this ligand is already being incorporated into clinical trial research, validation studies such as this paper are absolutely critical to evaluate the potential usefulness of this ligand as a reliable marker of human brain tau lesions. In an attempt to advance towards that goal, we applied [F-18]-MK-6240 phosphor screen and high resolution autoradiography to the study of a series of autopsy samples from individuals with a definitive diagnosis of AD, PiD, PSP, CBD, CTE, CAA, FTLD-TDP-43, DLB, and control brains free of neurodegenerative pathology. Our results confirmed that while [F-18]-MK-6240 avidly bound to PHF-tangle containing slices from AD brains, it did not bind to a significant extent to tau-containing lesions in slices from non-AD tauopathy brains, suggesting that this tracer has higher affinity and selectivity for PHF-tau over tau aggregates with a primarily straight filament ultrastructure, and thus raising reasonable doubts about the potential value of this ligand as a biomarker of tau pathology in non-AD tauopathies. The regional and laminar autoradiographic patterns of distribution of [F-18]-MK-6240, as revealed by the combination of autoradiography using a fine grain nuclear emulsion and immunohistochemistry, closely matched those of classic PHF-tangles in AD brains [1, 18]. Using this method, we confirmed that [F-18]-MK-6240-labeled lesions were NFT, suggesting that these lesions are the main pathological substrate of [F-18]-MK-6240 binding. The microscopic examination of diffuse plaques, CAA, α-synuclein and TDP-43 aggregates confirmed the absence of detectable [F-18]-MK-6240 binding to these lesions, favoring the relative selectivity of [F-18]-MK-6240 for NFT over β-amyloid plaques and other abnormal protein aggregates with a β-pleated sheet conformation.

Our data also establish that MK-6240 is not fully selective for PHF-tau deposits. Similarly to AV-1451, MK-6240 exhibits strong off-target binding to neuromelanin- and melanin-containing cells including pigmented neurons in the substantia nigra (regardless of the presence or absence of nigral tau pathology), leptomeningeal melanocytes, metastatic melanoma and retinal pigment epithelium, with some weaker off-target binding to brain hemorrhages as well. This is something relevant for the correct interpretation of [F-18]-MK-6240 in vivo imaging depending for example on the relative abundance and distribution of leptomeningeal melanocytes across different individuals [10], the possibility of focal artifactual increases in the density of these cells due to regional cortical atrophy, or the presence of concomitant brain hemorrhagic lesions.

One of the first generation tau PET tracers, THK-5351, has been recently found to demonstrate high binding affinity to MAO-B [13, 24], seriously compromising its value as a tau-specific tracer and increasing the need for alternative tau-specific imaging agents. To date, studies on potential non-specific binding of AV-1451 to MAO enzymes are scarce and have yielded conflicting results. A recent study by Vermeiren and colleagues suggested that H3-AV-1451 binds with similar nanomolar affinity to tau fibrils and MAO-A and B enzymes in brain homogenates isolated from AD or PSP patients as well as those devoid of tau pathology [30]. Merck’s researchers also reported high affinity displacement of 3H-AV-1451 binding, but not of 3H-MK-6240, in some non-AD brain homogenates in the presence of selective MAO-A inhibitor clorgyline. By contrary, Hansen and colleagues found that MAO-B inhibitors did not block in vivo [F-18]-AV-1451 binding in a series of 16 of 27 PD patients receiving MAO-B inhibitors at the time of scan [12]. In agreement with these results, Lemoine et al. reported that AV-1451 shows ten times lower affinity to MAO-B when compared to THK-5351 in in vitro assays [17]. Consistent with these observations, our data derived from [F-18]-MK-6240 and [F-18]-AV-1451 autoradiography experiments in the presence of selective MAO-A and MAO-B inhibitors point to a low binding affinity of both tracers for MAO enzymes. Studies using the specific enzymatic inhibitors do not exclude interaction of MK-6240 with MAO isoforms at regions removed from the active site. The discrepancies among the different studies could stem from the different techniques and isotope labeling used in each case e.g. in vitro binding assays in brain homogenates vs. autoradiography assays in tissue slices, and labeling of the tau tracer with H-3 vs. F-18. Our previous experience with H-3-AV-1451 in vitro binding assays in brain homogenates suggested that the signal-to background noise ratio when using this method was relatively low compared to [F-18]-AV-1451 autoradiographic techniques; something that could be due, at least in part, to off-target binding to sample components such as blood.

In conclusion, all together our results show that MK-6240 exhibits a nearly identical binding profile to AV-1451 holding promise as a potential surrogate marker for the in vivo detection of neurofibrillary tangles in AD, while still having various forms of off-target binding that need to be considered when interpreting in vivo imaging findings. The utility of MK-6240 for the reliable in vivo detection of tau aggregates in non-AD tauopathies, however, seems very limited. Both, MK-6240 and AV-1451, as opposed to other tracers like THK-5351, seem to exhibit relatively low binding affinity for MAO enzymes. Future imaging-pathological correlation studies on postmortem material from patients scanned while alive will provide additional information on the utility of these two tracers for the reliable quantification of NFT burden in AD and disease progression tracking by in vivo neuroimaging, as well as their potential usefulness when testing therapeutic approaches aimed at decreasing or halting the progression of tau aggregation in AD.