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).
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.
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.
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
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