Identification of the allosteric P2X7 receptor antagonist [11C]SMW139 as a PET tracer of microglial activation

Abstract

The P2X₇ receptor plays a significant role in microglial activation, and as a potential drug target, the P2X₇ receptor is also an interesting target in positron emission tomography. The current study aimed at the development and evaluation of a potent tracer targeting the P2X₇ receptor, to which end four adamantanyl benzamide analogues with high affinity for the human P2X₇ receptor were labelled with carbon-11. All four analogues could be obtained in excellent radiochemical yield and high radiochemical purity and molar activity, and all analogues entered the rat brain. [¹¹C]SMW139 showed the highest metabolic stability in rat plasma, and showed high binding to the hP2X₇ receptor in vivo in a hP2X₇ receptor overexpressing rat model. Although no significant difference in binding of [¹¹C]SMW139 was observed between post mortem brain tissue of Alzheimer's disease patients and that of healthy controls in in vitro autoradiography experiments, [¹¹C]SMW139 could be a promising tracer for P2X₇ receptor imaging using positron emission tomography, due to high receptor binding in vivo in the hP2X₇ receptor overexpressing rat model. However, further investigation of both P2X₇ receptor expression and binding of [¹¹C]SMW139 in other neurological diseases involving microglial activation is warranted.

Figure 1

Synthesis of adamantanyl benzamide precursors 5–8. Reagents and conditions: (a) BBr₃, CH₂Cl₂, 0 °C to rt, 48 h, quant. (b) PyBOP, DiPEA, CH₂Cl₂, rt, 18 h, 73% (6), 77% (7), 79% (8).

Figure 2

Carbon-11 methylation of benzamide analogues 1–3 and [¹¹C]SMW139.

Figure 3

Ex vivo biodistribution following intravenous administration of 13–23 MBq of [¹¹C]1–3 and [¹¹C]SMW139 in healthy male Wistar rats (n = 3 per tracer per time point). Good initial brain uptake was observed for all four analogues. Data are expressed as standardized uptake value (SUV) ± standard error of the mean (SEM).

Figure 4

Autoradiograms of transversal cryosections of rAAV-3flag-hP2X₇R vector injected rat brain. (A) Increased [¹¹C]SMW139 binding is observed in rAAV_3flag-hP2X₇R injected striatum (left on image). (B) [¹¹C]SMW139 binding in rAAV_3flag-hP2X₇R striatum is blocked (79 ± 10%, n = 2) by co-incubation with A-740003 (10 µM). (C) [¹¹C]SMW139 binding in rAAV_3flag-hP2X₇R striatum is blocked (93 ± 1%, n = 2) by co-incubation with JNJ-47965567 (10 µM).

Figure 5

Immunohistochemical staining for P2X₇R of transversal cryosections of rAAV-3flag-hP2X₇R vector injected rat brain. Left: rAAV_3flag-eGFP injected striatum with magnification in upper left corner. Right: rAAV_3flag-hP2X₇R injected striatum with magnification in upper right corner.

Figure 6

PET imaging with [¹¹C]SMW139 in rAAV_3flag-hP2X₇R rat model. (A) Representative PET-CT image (summed images 0–60 min) of [¹¹C]SMW139 showing increased uptake in rAAV_3flag-hP2X₇R injected striatum compared with rAAV_3flag-eGFP (control vector) injected striatum (n = 3). (B) Time-activity curves (TACs) of rAAV_3flag-hP2X₇R injected striatum and contralateral (rAAV_3flag-eGFP injected) striatum. Ellipsoid regions of interest (ROIs) were defined within striatum (dimensions: x = 3.4 mm, y = 3.6 mm, z = 2.9 mm) (C) Representative PET-CT image (summed images 0–60 min) of [¹¹C]SMW139 after pre-treatment with JNJ-47965567 (30 mg·kg⁻¹ s.c., 45 min prior to tracer injection). (D) TACs of rAAV_3flag-hP2X₇R injected and contralateral striatum after pre-treatment. Ellipsoid ROIs were defined within striatum (dimensions: x = 3.4 mm, y = 3.6 mm, z = 2.9 mm).

Figure 7

Autoradiograms of post mortem human brain tissue (temporal cortex). (A) Total binding of [¹¹C]SMW139 (28 nM). Upper row: AD patients, lower row: non-neurological controls (CTRL). Binding of [¹¹C]SMW139 does not differ significantly between AD patients and controls. Higher tracer binding was observed in white matter compared with grey matter. (B) Binding of [¹¹C]SMW139 could be fully blocked with 10 µM of JNJ-47965567 in both AD patients (upper row) and non-neurological age-matched controls (lower row).

Figure 8

Representative images of immunohistochemical stainings on post mortem brain material of AD patients and non-neurological controls. Overall, staining for all markers is more pronounced in AD patients compared with non-neurological controls (CTRL), particularly in grey matter (GM). Staining for P2X₇R is slightly, but significantly increased in both GM and WM of AD patients vs. CTRL. Scale bars indicate 100 µm, except in MHC-II images (50 µm).

Figure 9

Quantification of immunohistochemical staining in human post mortem brain tissue. (A) Quantification of immunohistochemical staining in grey matter depicted per marker (Aβ, tau, Iba1, MHC-II, CD68 and P2X₇R). Levels of pathological markers Aβ and tau were higher in AD patients (red circles) than in controls (blue diamonds) Levels of microglial markers Iba1, MHC-II, CD68 and P2X₇R were significantly higher in AD patients compared with controls. *p < 0.05, **p < 0.01. (B) Quantification of immunohistochemical staining in white matter depicted per marker (Aβ, tau, Iba1, MHC-II, CD68 and P2X₇R). Levels of microglial markers MHC-II, CD68 and P2X₇R were significantly higher in AD patients (red circles) compared with controls (blue diamonds). *p < 0.05, **p < 0.01.

Figure 10

Quantification of binding of [¹¹C]SMW139 in autoradiography experiments with human post mortem brain tissue. Both in grey (left) and white (right) matter, no significant differences were observed between AD patients (red circles) and non-neurological controls (blue squares).