A novel antioxidant ergothioneine PET radioligand for in vivo imaging applications

Abstract

Ergothioneine (ERGO) is a rare amino acid mostly found in fungi, including mushrooms, with recognized antioxidant activity to protect tissues from damage by reactive oxygen species (ROS) components. Prior to this publication, the biodistribution of ERGO has been performed solely in vitro using extracted tissues. The aim of this study was to develop a feasible chemistry for the synthesis of an ERGO PET radioligand, [11C]ERGO, to facilitate in vivo study. The radioligand probe was synthesized with identical structure to ERGO by employing an orthogonal protection/deprotection approach. [11C]methylation of the precursor was performed via [11C]CH3OTf to provide [11C]ERGO radioligand. The [11C]ERGO was isolated by RP-HPLC with a molar activity of 690 TBq/mmol. To demonstrate the biodistribution of the radioligand, we administered approximately 37 MBq/0.1 mL in 5XFAD mice, a mouse model of Alzheimer’s disease via the tail vein. The distribution of ERGO in the brain was monitored using 90-min dynamic PET scans. The delivery and specific retention of [11C]ERGO in an LPS-mediated neuroinflammation mouse model was also demonstrated. For the pharmacokinetic study, the concentration of the compound in the serum started to decrease 10 min after injection while starting to distribute in other peripheral tissues. In particular, a significant amount of the compound was found in the eyes and small intestine. The radioligand was also distributed in several regions of the brain of 5XFAD mice, and the signal remained strong 30 min post-injection. This is the first time the biodistribution of this antioxidant and rare amino acid has been demonstrated in a preclinical mouse model in a highly sensitive and non-invasive manner.

Improved synthesis of an ergothioneine PET radioligand for imaging oxidative stress in Alzheimer’s disease

“…Next, reductive methylation of a primary amine with aldehyde and sodium cyanoborohydride produced intermediate 4 with good yield. The incorporation of the thioketone into the imidazole ring and protection of the active functional groups in imidazole ring with Boc groups were performed as described in the past to provide precursor 6 [6].…”

“…Basically, to set the stage for the crucial incorporation of the thiol group onto imidazole, the amino and carboxyl groups of histidine were blocked with acidic labile moieties, while the imidazole amine was protected with an Fmoc group. We previously reported that the amine‐protecting Boc groups could be removed using TFA in the presence the t ‐butyl ester at −78 °C [6]. This reaction confers good yield, albeit only with stringent operations and attention to detail.…”

“…It has been reported that ERGO can prevent cell and tissue damage, a key contributor to aging, by protecting against free radicals and oxidative stress [3–5]. The chemical structure of ERGO is similar to that of histidine but with the presence of a thione group on the imidazole ring [6]. It is reported that ERGO protects against cytotoxicity by scavenging singlet oxygen, hydroxyl radicals [7–9], hypochlorous acid (HOCl), and peroxyl radicals [9,10], and acting as an inhibitor of iron or copper ion‐dependent generation of hydroxyl radicals from hydrogen peroxide [7].…”

“…The reproducibility of this design is the real impetus behind this work. This article describes an improved synthesis with an overall yield of 24% starting from the methyl ester to the precursor, compared to 14% as reported in the past [6].…”

The ergothioneine transporter (ETT): substrates and locations, an inventory

“…Even 30 min after injection of 11 C‐labeled ET (PET radioligand) into the tail of Alzheimer's model mice, relatively little was found in the brain (external fig. 6) [99]. Uptake in the kidney was 12‐fold higher and in the liver 8‐fold higher.…”