Subtle changes in neurotransmission that occur during Alzheimer’s disease have been tough to track in living people. A new positron emission tomography tracer may change that. In the January 12 Science Translational Medicine, researchers led by Wenping Li at Merck described MK-6884, a small carbon-11-labelled molecule that detects the M4 subtype of muscarinic cholinergic receptors. The tracer nuzzles into an allosteric pocket, latching on more tightly in the presence of higher acetylcholine concentrations, such as when monkeys and people were given the acetylcholinesterase inhibitor donepezil. MK-6884 opens the door for researchers to measure cholinergic drug efficacy and the neuropathological loss of acetylcholine receptors in AD and other diseases.

  • PET tracer MK-6884 binds to an allosteric site on M4 receptors.
  • People with low MMSE scores appear to bind less ligand.
  • MK-6884 may track loss of M4 receptor activity or drug efficacy.

“This tracer will enable us to study muscarinic receptor subtype specificity in living people,” Allan Levey of Emory University School of Medicine in Atlanta told Alzforum. Elliott Mufson, Barrow Neurological Institute, Phoenix, agreed. “Together, the data derived using this M4 PET tracer has the potential to contribute to our understanding of the selective vulnerability of select neurotransmitter systems and their association with cognitive decline in both neurological and neuropsychiatric disorders,” he wrote to Alzforum.

As AD progresses and basal forebrain cholinergic neurons succumb to the disease, the concentration of neurotransmitter acetylcholine wanes (Apr 2020 conference news; reviewed by Hampel et al., 2018). To study cholinergic receptor expression in living people, scientists have recently developed PET tracers that light up either all subtypes of muscarinic receptors or just the M1 variant (Rowe et al., 2021; Naganawa et al., 2021). 

Among the five receptor subtypes, researchers are particularly interested in M4. That's because its expression corresponds to neuropsychiatric symptoms, and enhancing its function with allosteric modulators improves memory, at least in mice (Gould et al., 2018; Bubser et al., 2014; Koshimizu et al., 2012). 

Aiming to study M4 receptors, and ultimately psychiatric symptoms, in AD, Li and colleagues had developed MK-6884 as an M4-positive allosteric modulator (Tong et al., 2020). It crossed the blood-brain barrier in monkeys and a carbon-11-labelled version brightly lit up the striatum and less so the cortex, in keeping with expression patterns of these receptors in primates (Levey, 1996; Volpicelli and Levey, 2004).

Meet MK-6884. When labeled with 11C, this molecule binds M4 muscarinic receptors. [Courtesy of Li et al., Science Translational Medicine, 2022.]

In this study, the scientists used [11C]MK-6884 to probe M4 receptor activity in the brain. The tracer is a cooperative positive allosteric modulator, meaning it increases binding of the M4 receptors’ natural ligand, acetylcholine, and acetylcholine in turn tightens MK-6884 binding. To test this cooperativity in animals, first author Li drove up acetylcholine concentrations in four rhesus macaques by injecting donepezil, then injecting MK-6884. Indeed, tracer binding in the animals’ striata rose dose-dependently with the acetylcholinesterase inhibitor.

Would MK-6884 bind similarly in the human brain? The researchers intravenously injected the tracer into seven healthy volunteers aged 55 to 85, who then immediately had PET scans. Ligand uptake was strongest in the striatum, moderate in the cortex and hippocampus, and minimal in the cerebellum (see image below). This is consistent with uptake of the non-subtype-specific and M1-specific muscarinic PET tracers. “The distribution is exactly what you would expect based on M4 RNA and protein expression patterns in the brain as well,” Levey said.

MK-6884 in the Human Brain. In a healthy old person, MK-6884 mostly bound in the striatum, but also in cortical regions. It passed right through the cerebellum. [Courtesy of Li et al., Science Translational Medicine, 2022.]

What about the effect of donepezil in people? The same healthy participants took 5 mg each day for one week, then 10 mg daily for two weeks before undergoing another PET scan. These are the same doses an AD patient would take, but the titration was quicker. As in monkeys, striatal uptake of MK-6884 rose 23 percent from baseline. Donepezil had no effect on ligand binding in the frontal or temporal cortices. It remains to be seen why these regions respond differently to the cholinesterase inhibitor, but the authors noted a much higher range of binding in the striatum, and they think this might be why they could detect a small allosteric effect there.

What did tracer uptake look like in the AD brain? Compared to the healthy adults taking donepezil, 10 people with moderate to severe AD bound 18 percent less ligand in their striata and 45 percent less in their temporal cortices. All were already taking donepezil or rivastigmine, another cholinesterase inhibitor approved and widely prescribed for AD. 

The authors believe that either people with AD have fewer M4 receptors, or that lower acetylcholine concentrations result in less allosteric modulation of the receptors and hence weaker tracer binding. Agneta Nordberg, Karolinska Institutet, Stockholm, agreed. “The different effects of donepezil treatment on MK-6884 binding probably reflect different regulatory mechanisms involving presynaptic M4 receptors in normal and diseased brains,” she wrote to Alzforum.

MK-6884 binding was also lower in the parietal and occipital cortices of the AD cases than in controls. Since muscarinic receptors in those areas contribute to visuospatial processing, this might explain deficits in such processing in AD, suggested the authors.

Given the role of M4 receptors in cognition, could MK-6884 binding be used as a marker of cognitive decline? The researchers correlated each person’s MMSE scores with their PET scan. Though people with lower scores bound less ligand in their brains, the correlation was not significant (see image below). The authors chalked this up to the small sample size and binding heterogeneity.

Waning in Alzheimer’s. Compared to one healthy control (top left), five people in their 60s and 70s with AD bound less MK-6884 in the brain. Binding trended weaker with lower MMSE scores. [Courtesy of Li et al., Science Translational Medicine, 2022.]

Ideally, PET tracers should be stable until the scan has completed, but this one might be quickly metabolized by the body. Tharick Pascoal, University of Pittsburgh, noted that the ratio of positrons detected in the brain to those in the plasma changed over time, suggesting that a metabolite of MK-6884 may have crossed the BBB and gotten trapped in the brain. “This warrants caution for quantification in future clinical studies,” he wrote.

What else could this PET tracer tell researchers about AD? Levey thinks MK-6884 can track changes in M4 receptors over time. Mufson agreed and suggested using the tracer to compare changes in muscarinic and nicotinic cholinergic receptor activity during AD progression.

MK-6884 could also track cholinergic response to acetylcholinesterase inhibitors or other treatments or even help clinicians titrate patients to the right dose. “This ligand may be useful for visualizing the effect of anti-amyloid and tau drugs on M4 receptor activity and cognition in patients with AD, particularly during the preclinical or prodromal phases,” Mufson wrote.

In 2019, Merck licensed MK-6884 to Enigma Biomedical Group, a Canadian company that partners with academic and pharmaceutical labs to develop imaging agents. They plan to study the tracer in clinical trials.—Chelsea Weidman Burke


  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.

  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


    . 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.

    . 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.

    . 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.

    . 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.

  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.

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News Citations

  1. Does Alzheimer’s Start in the Heart of the Cholinergic System?

Paper Citations

  1. . The cholinergic system in the pathophysiology and treatment of Alzheimer's disease. Brain. 2018 Jul 1;141(7):1917-1933. PubMed.
  2. . PET Imaging of brain muscarinic receptors with 18F-Fluorobenzyl-Dexetimide: A first in human study. Psychiatry Res Neuroimaging. 2021 Oct 30;316:111354. Epub 2021 Aug 8 PubMed.
  3. . First-in-Human Assessment of 11C-LSN3172176, an M1 Muscarinic Acetylcholine Receptor PET Radiotracer. J Nucl Med. 2021 Apr;62(4):553-560. Epub 2020 Aug 28 PubMed.
  4. . Cognitive enhancement and antipsychotic-like activity following repeated dosing with the selective M4 PAM VU0467154. Neuropharmacology. 2018 Jan;128:492-502. Epub 2017 Jul 17 PubMed.
  5. . Selective activation of M4 muscarinic acetylcholine receptors reverses MK-801-induced behavioral impairments and enhances associative learning in rodents. ACS Chem Neurosci. 2014 Oct 15;5(10):920-42. Epub 2014 Aug 19 PubMed.
  6. . M4 muscarinic receptor knockout mice display abnormal social behavior and decreased prepulse inhibition. Mol Brain. 2012 Apr 2;5:10. PubMed.
  7. . Discovery of [11C]MK-6884: A Positron Emission Tomography (PET) Imaging Agent for the Study of M4Muscarinic Receptor Positive Allosteric Modulators (PAMs) in Neurodegenerative Diseases. J Med Chem. 2020 Mar 12;63(5):2411-2425. Epub 2020 Mar 3 PubMed.
  8. . Muscarinic acetylcholine receptor expression in memory circuits: implications for treatment of Alzheimer disease. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13541-6. PubMed.
  9. . Muscarinic acetylcholine receptor subtypes in cerebral cortex and hippocampus. Prog Brain Res. 2004;145:59-66. PubMed.

External Citations

  1. Enigma Biomedical Group

Further Reading


  1. . Discovery of a Novel Muscarinic Receptor PET Radioligand with Rapid Kinetics in the Monkey Brain. ACS Chem Neurosci. 2018 Feb 21;9(2):224-229. Epub 2017 Nov 13 PubMed.
  2. . PET Imaging Estimates of Regional Acetylcholine Concentration Variation in Living Human Brain. Cereb Cortex. 2021 May 10;31(6):2787-2798. PubMed.

Primary Papers

  1. . 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.