Li W, Wang Y, Lohith TG, Zeng Z, Tong L, Mazzola R, Riffel K, Miller P, Purcell M, Holahan M, Haley H, Gantert L, Hesk D, Ren S, Morrow J, Uslaner J, Struyk A, Wai JM, Rudd MT, Tellers DM, McAvoy T, Bormans G, Koole M, Van Laere K, Serdons K, de Hoon J, Declercq R, De Lepeleire I, Pascual MB, Zanotti-Fregonara P, Yu M, Arbones V, Masdeu JC, Cheng A, Hussain A, Bueters T, Anderson MS, Hostetler ED, Basile AS. The PET tracer [11C]MK-6884 quantifies M4 muscarinic receptor in rhesus monkeys and patients with Alzheimer's disease. Sci Transl Med. 2022 Jan 12;14(627):eabg3684. PubMed.

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  1. Li and colleagues describe the discovery and translation of the PET tracer [11C]MK-6884 to visualize M4 muscarinic cholinergic receptors in the brain of rhesus monkeys and patients with Alzheimer’s disease (AD). Since most studies of alterations to muscarinic receptor activity in human brain used single photon emission computed tomography (SPECT), this new ligand provides an additional imaging tool for the field of AD research.

    The development of this novel M4 PET tracer has the potential to improve our understanding of brain M4 binding dysfunction in several diseases that display cholinergic basocortical degeneration, including Parkinson’s disease, Lewy body disease, AD as well as neuropsychiatric disorders.

    This PET tracer can also be used to evaluate the efficacy of drugs that modulate M4 receptors, either independently or in conjunction with other cholinergic treatment approaches (i.e., anticholinesterases) that remain a mainstay for the treatment of AD. This ligand may be useful in visualizing the effect that anti-amyloid and/or tau drugs have upon M4 receptor activity and cognition in patients with AD, particularly during the preclinical or prodromal phases of this disease.

    It will also allow for detailed imaging studies comparing changes in muscarinic compared to nicotinic receptor activity, particularly the alpha 7 nicotinic receptor that is dysregulated in cholinergic neurons of the basal forebrain and their cortical projection sites during the progression of AD. This novel ligand could be used in aging and lesion-induced studies of the cholinergic degeneration in a primate model of AD.

    Together, the data derived using this M4 PET tracer has the potential to contribute to our understanding of the selective vulnerability of select neurotransmitter system and their association with cognitive decline in both neurological and neuropsychiatric disorders.

    View all comments by Elliott Mufson

  2. This work provides a new tool for imaging the cholinergic machinery in the context of Alzheimer’s disease (AD). Specifically, the authors reported the validation, from non-human primates to human AD patients, of [11C]MK-6884 – a recently developed PET radiotracer targeting the acetylcholine (ACh) muscarinic receptor M4 (M4R). This tracer shows potential to evaluate the target engagement of drugs selective for the M4R.

    The M4R is associated with neuropsychiatric symptoms (NPS), such as hallucinations and delusions, mostly diagnosed in schizophrenia and, less frequently, in AD. It is worth mentioning that NPS and the underlying brain mechanisms in AD might differ from those observed in schizophrenia.

    In this study, the authors described the successful radiosynthesis of [11C]MK-6884 from the N-alkylation of the parent lactam using [11C]methyl iodide and obtained high radiochemical purity (> 95%) and good molar activity (39 to 741 GBq/µmol). Furthermore, a tritiated version of MK-6884 ([3H]MK-6884) was developed for measuring the allosteric sites on the M4R in different regions of the mammalian brain. [3H]MK-6884 had great Kd values of 0.9 nM and 1.2 nM in rhesus monkeys and humans, respectively. However, the moderate to low receptor density (Bmax) in the mammalian brain might be an issue (rhesus monkey = 13 nM, human = 7 nM).

    Previous studies on PET radiotracer development strongly suggest that the ratio between Bmax/Kd should be at least 10 to provide a good signal-to-noise ratio in vivo (Watabe et al., 2000). The saturation binding assays of [3H]MK-6884 demonstrated a ratio below the limit for human brain (5.8), but a ratio satisfactory for rhesus monkeys (14.4), which could affect the PET radiotracer sensitivity in vivo.

    Moreover, the Scatchard plots of [3H]MK-6884 binding (Figure 2D) suggest at least two binding sites for the PET radiotracer in the human striatum, which we believe will be interesting to investigate further. Additional in-vitro assays are needed to explore potential off-target binding to amyloid and tau. Further, brain autoradiography studies in postmortem AD brain tissue and receptor binding studies in P2 fraction (as previously demonstrated by our group) would have been useful to provide the full picture regarding the binding properties/mechanisms of [3H]MK-6884 in the human brain (Marutle et al., 1998).

    The PET imaging biodistribution of [11C]MK-6884 was evaluated in seven healthy subjects (55-85 ys) and 10 age-matched AD patients with moderate to severe dementia (<20 MMSE). Corroborating with in vitro findings, the authors showed high binding potential (BP) of [11C]MK-6884 in the striatum, a region well-known for high levels of ACh, choline acetyl transferase (ChAT), and acetylcholinesterase (AChE) activity and great mRNA/protein expression of M4Rs - in both rhesus monkey and human brain (Pancani et al., 2014).

    Nevertheless, [11C]MK-6884 had comparable BP in the human striatum and cortex (Figure 5C), in contrast to previous findings showing lesser amounts of M4Rs in the cortex compared to striatum4. On the other hand, [11C]MK-6884 demonstrated greater BP in the striatum than in cortex and hippocampus of rhesus monkeys. The Bmax/Kd ratio of 5.8 for human and 14.4 for monkey brains could explain the greater behavior of [11C]MK-6884 to image the M4Rs in non-human primates.

    In addition, evaluation of metabolites in the plasma following [11C]MK-6884 administration highlighted the presence of at least four metabolic by-products. The authors suggest that due to high polarity observed by HPLC metabolite analysis, these compounds are unlikely to penetrate the blood-brain barrier (BBB). Nevertheless, a further and careful analysis of brain metabolites should be considered before ruling out the possible interference in clinical PET imaging analysis of M4R using [11C]MK-6884.

    To explore the sensitivity of [11C]MK-6884 to detect M4R in AD, the authors compared healthy subjects and AD patients who received donezepil. It has been shown that donezepil increased [11C]MK-6884 BP in the striatum of healthy individuals but didn’t affect the BP in the cortical regions. Yet, the [11C]MK-6884 binding was decreased in both cortex and striatum of AD patients treated with donezepil. This difference, observed in AD and healthy subjects, could be explained by different regulatory mechanisms involving the presynaptic M4R in the cholinergic synapse, after inhibition of AChE activity.

    Furthermore, a crucial point to determine the applicability of [11C]MK-6884 to estimate the target engagement of drugs selective for M4R in AD, should be the inclusion of an additional group of AD patients who didn’t receive donezepil treatment. With this, it will be possible to evaluate if this reduced binding of [11C]MK-6884 observed in moderate to severe AD patients was due to treatment or to different M4R regulatory mechanisms and disturbances of the cholinergic acetylcholine system in the AD brain. Moreover, it would be interesting to investigate the influence of antagonist drugs such as scopolamine (used for motion sickness) or diphenhydramine (most common antihistamine used for allergy) on the binding behavior MK-6884.

    Overall, the study presented by Li et al. is of great interest, since it provides a new tool to study the cholinergic machinery in human brain and in neurodegenerative disorders. We believe that developing innovative PET tracers is the key for boosting the identification of new therapeutic targets in AD.

    Co-authored by Igor Fontana

    References:

    Li W, Wang Y, Lohith TG, Zeng Z, Tong L, Mazzola R, Riffel K, Miller P, Purcell M, Holahan M, Haley H, Gantert L, Hesk D, Ren S, Morrow J, Uslaner J, Struyk A, Wai JM, Rudd MT, Tellers DM, McAvoy T, Bormans G, Koole M, Van Laere K, Serdons K, de Hoon J, Declercq R, De Lepeleire I, Pascual MB, Zanotti-Fregonara P, Yu M, Arbones V, Masdeu JC, Cheng A, Hussain A, Bueters T, Anderson MS, Hostetler ED, Basile AS. The PET tracer [11C]MK-6884 quantifies M4 muscarinic receptor in rhesus monkeys and patients with Alzheimer's disease. Sci Transl Med. 2022 Jan 12;14(627):eabg3684. PubMed.

    Watabe H, Endres CJ, Breier A, Schmall B, Eckelman WC, Carson RE. Measurement of dopamine release with continuous infusion of [11C]raclopride: optimization and signal-to-noise considerations. J Nucl Med. 2000 Mar;41(3):522-30. PubMed.

    Marutle A, Warpman U, Bogdanovic N, Nordberg A. Regional distribution of subtypes of nicotinic receptors in human brain and effect of aging studied by (+/-)-[3H]epibatidine. Brain Res. 1998 Aug 10;801(1-2):143-9. PubMed.

    Pancani T, Bolarinwa C, Smith Y, Lindsley CW, Conn PJ, Xiang Z. M4 mAChR-mediated modulation of glutamatergic transmission at corticostriatal synapses. ACS Chem Neurosci. 2014 Apr 16;5(4):318-24. Epub 2014 Feb 27 PubMed.

    View all comments by Igor Fontana

  3. The novel PET tracer shows some favorable characteristics, such as relatively fast brain penetration and kinetics, good affinity with a dissociation constant within nanomolar range, selectivity, and brain uptake suitable for imaging humans with distribution resembling the expected to M4Rs. The fact that results in monkeys and humans (e.g., instability in total distribution volume over time) suggest the possibility of a brain-penetrant metabolite warrants caution for the quantification of this tracer in clinical studies. Future work should clarify the magnitude of the possible impact of the metabolite on accurate brain quantification.

    The use of this tracer in larger studies across the AD spectrum, in combination with other biomarkers, could help to elucidate dynamic associations of cholinergic system dysfunction with disease stage, Aβ and tau accumulations, glial cell abnormalities, etc. The small number of subjects and their previous use of acetylcholine esterase inhibitors limit the clinical-pathophysiological interpretation of the results in the present study.

    In summary, this is an important study, providing an informative validation of a new PET tracer that can be used to assess target engagement of allosteric modulators of M4Rs, and to potentially clarify pathohistological associations in future clinical studies.

    View all comments by Tharick Pascoal

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  1. New PET Tracer Binds Muscarinic Acetylcholine Receptors