Potential for in vivo visualization of intracellular pH gradient in the brain using PET imaging

Intracellular pH is a valuable index for predicting neuronal damage and injury. However, no PET probe is currently available for monitoring intracellular pH in vivo. In this study, we developed a new approach for visualizing the hydrolysis rate of monoacylglycerol lipase, which is widely distributed in neurons and astrocytes throughout the brain. This approach uses PET with the new radioprobe [¹¹C]QST-0837 (1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-phenyl-1H-pyrazol-3-yl)azetidine-1-[¹¹C]carboxylate), a covalent inhibitor containing an azetidine carbamate skeleton for monoacylglycerol lipase. The uptake and residence of this new radioprobe depends on the intracellular pH gradient, and we evaluated this with in silico, in vitro and in vivo assessments. Molecular dynamics simulations predicted that because the azetidine carbamate moiety is close to that of water molecules, the compound containing azetidine carbamate would be more easily hydrolyzed following binding to monoacylglycerol lipase than would its analogue containing a piperidine carbamate skeleton. Interestingly, it was difficult for monoacylglycerol lipase to hydrolyze the azetidine carbamate compound under weakly acidic (pH 6) conditions because of a change in the interactions with water molecules on the carbamate moiety of their complex. Subsequently, an in vitro assessment using rat brain homogenate to confirm the molecular dynamics simulation-predicted behaviour of the azetidine carbamate compound showed that [¹¹C]QST-0837 reacted with monoacylglycerol lipase to yield an [¹¹C]complex, which was hydrolyzed to liberate ¹¹CO₂ as a final product. Additionally, the ¹¹CO₂ liberation rate was slower at lower pH. Finally, to indicate the feasibility of estimating how the hydrolysis rate depends on intracellular pH in vivo, we performed a PET study with [¹¹C]QST-0837 using ischaemic rats. In our proposed in vivo compartment model, the clearance rate of radioactivity from the brain reflected the rate of [¹¹C]QST-0837 hydrolysis (clearance through the production of ¹¹CO₂) in the brain, which was lower in a remarkably hypoxic area than in the contralateral region. In conclusion, we indicated the potential for visualization of the intracellular pH gradient in the brain using PET imaging, although some limitations remain. This approach should permit further elucidation of the pathological mechanisms involved under acidic conditions in multiple CNS disorders.