PET imaging of M4 muscarinic acetylcholine receptors in rhesus macaques using [11C]MK-6884: Quantification with kinetic modeling and receptor occupancy by CVL-231 (emraclidine), a novel positive allosteric modulator

Abstract

Stimulation of the M₄ muscarinic acetylcholine receptor reduces striatal hyperdopaminergia, suggesting its potential as a therapeutic target for schizophrenia. Emraclidine (CVL-231) is a novel, highly selective, positive allosteric modulator (PAM) of M4 muscarinic acetylcholine receptors i.e. acts as a modulator that increases the response of these receptors. First, we aimed to further characterize the positron emission tomography (PET) imaging and quantification performance of a recently developed M₄ PAM radiotracer, [¹¹C]MK-6884, in non-human primates (NHPs). Second, we applied these results to determine the receptor occupancy of CVL-231 as a function of dose. Using paired baseline-blocking PET scans, we quantified total volume of distribution, binding potential, and receptor occupancy. Both blood-based and reference region-based methods quantified M₄ receptor levels across brain regions. The 2-tissue 4-parameter kinetic model best fitted regional [¹¹C]MK-6884-time activity curves. Only the caudate nucleus and putamen displayed statistically significant [¹¹C]MK-6884 uptake and dose-dependent blocking by CVL-231. For binding potential and receptor occupancy quantification, the simplified reference tissue model using the grey cerebellum as a reference region was employed. CVL-231 demonstrated dose-dependent M₄ receptor occupancy in the striatum of the NHP brain and shows promise for further development in clinical trials.

Keywords: M4 mAChR; PAM; [11C]MK-6884; emraclidine; neuroimaging.

Figure 1.

Arterial whole-blood and plasma measurements and in vivo metabolism. (a) Representative radiochromatograms showing peaks corresponding to [¹¹C]MK-6884 parent and radiometabolites in plasma. Data are shown for the baseline M2.3 scan. (b) Time dependency of the %PP for each [¹¹C]MK-6884 scan. HPLC data for M1.3 were not acquired due to technical issues. (c) Representative whole-blood and parent-in-plasma time courses in SUV. The data for baseline and blocking scans are shown for the low (0.25 mg/kg) and high (1.7 mg/kg) doses of CVL-231 injected in monkey 1 and (d) Whole blood to plasma ratio of radioactivity concentrations. Abbreviations: BSLN-baseline, BLK-blocking, STD-standard, WB-whole blood, PP-parent in plasma, SUV-standard uptake value.

Figure 2.

PET SUV data. (a) PET SUV images of [¹¹C]MK-6884 at baseline and after each blocking dose of CVL-231 for monkey 1. The PET and MRI MEMPRAGE images are co-registered in the MRI NIMH macaque template (NMT) space using affine transformations. The red crosshairs are placed on the striatum area (characterized by the highest tracer uptake). PET SUV images are generated by summing dynamic frames over the 0–10 min (early phase reflecting tracer delivery) and 30–90 min (later phase more representative of tracer binding) time intervals and (b) SUV TACs in the sampled brain regions fitted with either the reversible one-tissue compartment model (1T2k) or the reversible two-tissue compartment model (2T4k) with the vascular contribution (V_f) fixed to a nominal value.

Figure 3.

Quantification of [¹¹C]MK-6884 binding by Logan DV analysis. (a) Scatter plot showing correlation between V_T estimates obtained by Logan DV and by the 2T4k kinetic model. A scan duration of 60 minutes was used for both methods. (b) Lassen plot based on V_T values for monkey 1 (top) and monkey 2 (bottom). Linear regressions are shown for each paired baseline-blocking study. (c) Bar plots showing regional V_T values for each paired baseline-blocking study in monkey 1 (top graph) and monkey 2 (bottom graph). Shaded areas indicate the 95%CI corresponding to the estimated V_ND, which were [1.18, 2.36] for monkey 1 and [1.46, 1.77] for monkey 2 and (d) Logan V_T parametric images of [¹¹C]MK-6884 binding at baseline and at each CVL-231 blocking dose in monkey 2. V_T and MEMPRAGE images were aligned to the MRI NIMH macaque template (NMT) space using affine transformations. The red crosshair is positioned on the striatum.

Figure 4.

Quantification of the striatal BP_ND by the simplified reference tissue (SRTM) models. (a) Representative baseline TACs fitted by the SRTM model using cerebellar grey matter as a reference tissue. (b) Striatal BP_ND values measured for each paired baseline-blocking study at baseline and at different blocking doses of CVL-231. BP_ND estimates were derived by SRTM using 60 minutes of data. (c) BP_ND parametric images of [¹¹C]MK-6884 at baseline and at each blocking dose of CVL-231 in monkey 2. Voxel-based SRTM2 modeling was performed using 60 minutes of data and k₂′ was constrained to values derived by ROI-based analysis within each individual scan. BP_ND and MEMPRAGE images were aligned to the MRI NIMH macaque template (NMT) space using affine transformations. The red crosshair is positioned on the striatum.

Figure 5.

Relationship between striatal M₄ mAChR occupancy by CVL-231 and CVL-231 mass dose (a) and CVL-231 plasma concentration (b) with data averaged across animals. Non-linear regression was performed to fit the data using a one-parameter E_max model based on a Hill function assuming a 100% maximal RO. The goodness of fit is indicated by R² statistics. The regression models provided estimates of ID₅₀ and IC₅₀, respectively. Corresponding standard errors and values for 95% confidence intervals (95%CI) are also indicated. The 95% confidence intervals are plotted using dashed lines.