PET ligands [18F]LSN3316612 and [11C]LSN3316612 quantify O-linked-β- N-acetyl-glucosamine hydrolase in the brain

Abstract

We aimed to develop effective radioligands for quantifying brain O-linked-β-N-acetyl-glucosamine (O-GlcNAc) hydrolase (OGA) using positron emission tomography in living subjects as tools for evaluating drug target engagement. Posttranslational modifications of tau, a biomarker of Alzheimer's disease, by O-GlcNAc through the enzyme pair OGA and O-GlcNAc transferase (OGT) are inversely related to the amounts of its insoluble hyperphosphorylated form. Increase in tau O-GlcNAcylation by OGA inhibition is believed to reduce tau aggregation. LSN3316612, a highly selective and potent OGA ligand [half-maximal inhibitory concentration (IC₅₀) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [³H]LSN3316612 imaged and quantified OGA in postmortem brains of rat, monkey, and human. The presence of fluorine and carbonyl functionality in LSN3316612 enabled labeling with positron-emitting fluorine-18 or carbon-11. Both [¹⁸F]LSN3316612 and [¹¹C]LSN3316612 bound reversibly to OGA in vivo, and such binding was blocked by pharmacological doses of thiamet G, an OGA inhibitor of different chemotype, in monkeys. [¹⁸F]LSN3316612 entered healthy human brain avidly (~4 SUV) without radiodefluorination or adverse effect from other radiometabolites, as evidenced by stable brain total volume of distribution (V_T) values by 110 min of scanning. Overall, [¹⁸F]LSN3316612 is preferred over [¹¹C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey.

Fig. 1.. Autoradiography of [³H]LSN3316612 binding to brain tissue.

[³H]LSN3316612 (5 nM) binding to OGA (left columns) or non-displaced bindings after thiamet G blocking (20 μM, right columns) in brain sections of (A) monkey (n = 1) and (B) human (n = 1). Brain regions are Cb = cerebellum; Cd = caudate nucleus; Hp = hippocampus; Hy = hypothalamus; Ns = substantia nigra; PFC = prefrontal cortex; Pu = putamen; and St = striatum.

Fig. 2.. Ex vivo distribution of LSN3316612 in rat brain.

(A) Tissue distribution and plasma concentrations of LSN3316612 over time. (B) Thiamet G blockade of LSN3316612 receptor occupancy in rats. LSN3316612 (10 μg/kg) was administered to rats intravenously. Data are mean ± SEM from n = 3–4 male rats per treatment group.

Fig. 3.. Radiosyntheses of [¹¹C]LSN3316612 and [¹⁸F]LSN3316612.

¹¹C-Labeling is by palladium-mediated [¹¹C]carbon monoxide insertion and [¹⁸F]-labeling by aromatic nucleophilic substitution with [¹⁸F]fluoride ion. Red indicates that ¹¹C and ¹⁸F are radioactive.

Fig. 4.. PET imaging of [¹¹C]LSN3316612 and [¹⁸F]LSN3316612 in monkey.

(A) Whole brain time-activity curves in a rhesus monkey administered [¹¹C]LSN3316612 at baseline and after preblocking of OGA with thiamet G (10 mg/kg, i.v.). (B) Summed brain PET images (0–120 min) after injection of [¹¹C]LSN3316612 at baseline (top row) and after preblocking of OGA with thiamet G (10 mg/kg, i.v.) (bottom row). (C) Whole brain time-activity curves in a rhesus monkey administered [¹⁸F]LSN3316612 at baseline and after preblocking of OGA with thiamet G (10 mg/kg, i.v.). (D) Summed brain PET images (0–120 min) after injection of [¹⁸F]LSN3316612 at baseline (top row) and after preblocking of OGA with thiamet G (10 mg/kg, i.v.) (bottom row). Images in (B, D) are: left, coronal scans; middle, sagittal scans; right, transaxial scans.

Fig. 5.. PET imaging of [¹⁸F]LSN3316612 in human subjects.

(A) Representative brain region PET time-activity curves in a healthy human subject (n = 8) administered [¹⁸F]LSN3316612 at baseline. (B) Representative derived parametric (V_T) human brain images (0–180 min) from the injection of [¹⁸F]LSN3316612 at baseline (bottom row) and the corresponding MRI images (top row) (left: coronal scans; middle: sagittal scans; right, transaxial scans). (C) Unchanged [¹⁸F]LSN3316612 in whole blood and in plasma after intravenous injection of [¹⁸F]LSN3316612 into a human subject. The plasma curve represents the radiometabolite-corrected arterial input function. (D) Normalized regional distribution volumes (V_T) as a function of duration of image acquisition after [¹⁸F]LSN3316612 injection at baseline in human subjects. V_T was normalized to values at 180 min.