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

Abstract

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.

Keywords: PET; hydrolysis; hypoxia; intracellular pH; monoacylglycerol lipase.

Graphical Abstract

Figure 1

The hypothesized mechanism for hydrolysis of the ¹¹C-labelled MAGL inhibitor containing an azetidine carbamate skeleton. (A) Proposed disassociation route for the ¹¹C-labelled MAGL inhibitor containing an azetidine carbamate moiety. (B) Diagram illustrating the effect of acidic sources on ¹¹CO₂ production.

Figure 2

MD simulations. (A) Chemical structures of compound-A and its MAGL complex. (B) Chart of the RMSD values for main chain atoms of complex-A in the MD simulations (5 ns) under neutral conditions. (C) 3D structures of complex-A. (D) Chemical structures of compound-P and its MAGL complex. (E) Chart of RMSD values for main chain atoms of complex-P in the MD simulations. (F) 3D structures of complex-P. (G) Overlaid structures of complex-A composed from MD simulations at pH 7 and pH 6. (H) Changes in the interaction of complex-A with water molecules between neutral (pH 7) and weakly acidic (pH 6) conditions. Spatial localization and interaction of the residue of complex-A with a water molecule at pH 7 (left). Changes in the interaction between the residue of complex-A and a water molecule at pH 6 (right). Hydrogen bonds in the complexes are shown as dotted lines. MD simulations were performed using 3D structures of MAGL (PDB: 6AX1).

Figure 3

In vitro  ¹¹CO₂ collection assay using rat brain homogenate. (A) Schematic illustration and compartments of the assay. (B) Radioactivity (percentage of incubation dose, %ID) derived from [¹¹C]QST-0837 or [¹¹C]HPPC in the three divided compartments (C1: unbound, C2: complex and C3: product). Data from three independent experiments for each radioprobe are shown as mean ± SD. NS: Not significant, ***P < 0.001 (two-way ANOVA with Bonferroni post hoc test). (C) Radioactivity (%ID) derived from [¹¹C]QST-0837 in the presence of different concentrations (0, 10, 30, 50 and 100 mM) of lactic acid (LA). Data from four independent experiments for each LA concentration are shown as mean ± SD. NS: Not significant, *P < 0.05, **P < 0.01 and ***P < 0.001 (two-way ANOVA with Bonferroni post hoc test). (D) The pH-response curve for ¹¹CO₂ production rate. The pH evoking half the maximal ¹¹CO₂ production was 5.3.

Figure 4

PET imaging in the brain of a healthy rat. (A) Representative 0–90 min summed PET–MRI images of [¹¹C]QST-0837. (B) Time–activity curves (n = 4) of [¹¹C]QST-0837 in the cerebral cortex, striatum, hippocampus, thalamus, pons/medulla and cerebellum. (C) Representative 0–90 min summed PET–MRI images of [¹¹C]HPPC. (D) Time–activity curves (n = 4) of [¹¹C]HPPC in the cerebral cortex, striatum, hippocampus, thalamus, pons/medulla and cerebellum. (E and F) Chase studies of [¹¹C]QST-0837 and [¹¹C]HPPC using an irreversible-type inhibitor for MAGL (JW642). (E) Time–activity curves (n = 3) of [¹¹C]QST-0837 in the brain of rat treated with JW642 (1 mg/kg, i.v.) 20 min after the scan started. (F) Time–activity curves (n = 3) of [¹¹C]HPPC in the brain of rat administered with JW642 (1 mg/kg, i.v.) 20 min after the scan started. ROIs were drawn in the cerebral cortex, striatum, hippocampus, thalamus, pons and cerebellum. Radioactivity is expressed as the SUV. Co, cerebral cortex; St, striatum; Hi, hippocampus; Th, thalamus; Po, pons/medulla; Ce, cerebellum.

Figure 5

PET imaging in the brain of a MCAO rat. (A) Diagrammatic reasoning of the clearance of radioactivity starting from [¹¹C]QST-0837. (B) Schematic of a compartment model for estimation of the kinetic parameters of the radioactivity. C_P: free radioactive compounds containing [¹¹C]QST-0837 and ¹¹CO₂ in the plasma; C1: free and non-specific binding of [¹¹C]QST-0837 in the brain tissue; C2: state under [¹¹C]complex-A; C3: ¹¹CO₂ as the final product of hydrolysis in the brain tissue; K₁: influx rate of [¹¹C]QST-0837; k₂: efflux rate of [¹¹C]QST-0837; k₃: association rate of [¹¹C]QST-0837 to MAGL; K_H: hydrolysis rate of MAGL; k₄: reuptake rate of ¹¹CO₂; k₅: efflux rate of ¹¹CO₂ from the brain side. (C) Representative PET images with [¹¹C]QST-0837 and TTC staining in the brains of rats subjected to MCAO for 3–4 h. PET images were generated by summing for 0–90 min after injection and scaling with the SUV. The brain slices were prepared after the PET scan and were stained using TTC. (D) Time–activity curves (TACs) of [¹¹C]QST-0837 in contralateral (Cont) and ipsilateral (Ips) sides. (E) Clearance rate of radioactivity in contralateral (Cont) and ipsilateral (Ips) sides. Radioactivity is expressed as the percentage of maximum radioactivity (SUV of 15 min). K_H was generated using a mono-exponential fitting. (F) PET images scaled with relative K_H value and immunofluorescent (IF) images reflecting hypoxic regions in the MCAO rat brains. Arrows indicate areas of intense reduction in K_H value and strong fluorescent signals. (G) Correlation plots between the maximum SUV value (SUV max) in TACs acquired from VOIs on brain slice adjacent from ROIs (shown in Supplementary Fig. 4) for quantification of immunohistochemical signals and K_H values estimated on their TACs. (H) Correlation plots between quantitative values for immunohistochemical signal in ROIs (shown in Supplementary Fig. 4) on three slides and K_H values estimated on TACs obtained from VOIs on brain slice adjacent from these ROIs. Relationship tests were conducted by a linear regression.