Development of Imaging Methods to Asses Glutamate Function at the NMDA ion channel

Objectives Interaction of the excitatory amino acid neurotransmitter, glutamate (GLU), with the N-Methyl-D-Aspartate (NMDA) receptor ion-channel may be adversely affected in Alzheimer ’s Disease (AD) brains. Our previous in vitro studies on postmortem AD human brain slices showed greater GLU induced hyper-activation using [3H]MK801 compared to normal controls, suggestive of anomalous functioning of the NMDA ion-channel in AD (Shah et al., 2015). Hence, the MK-801 binding site within the NMDA ion channel is a target of great interest for the development of therapeutic and imaging agents. In order to assess the function of the NMDA ion-channel in vivo, we report the development and evaluation of the amphiphilic fluorodeoxyglucamine, FDGluMK801, as a potential imaging agent for PET.

Methods FDGluMK801 was synthesized by reductive coupling of FDG with MK-801 using our recently published procedure (Baranwal et al., 2015). FDG (4mg) and MK-801 (5mg) were reacted in methanol and acetic acid, and then reduced using NaCNBH3. After successful product recovery, we also synthesized [18F]FDGluMK801, using the same reaction methods overall, and [18F]FDG. Binding affinity of FDGluMK801 was tested in mice brains, where brain homogenates in 5mM Tris/pH 7.4 buffer, each 100 µL, were incubated in 10 µM glutamate and glycine, and [3H]MK801. Non-specific (NS) binding was measured using standard MK-801 (10 µM). Tissue samples were incubated at 37°C for 2 hours, and were then filtered and counted. Human postmortem frontal cortex (FC) brain sections (AD, n=6, age 82-90, SP Stage C and normal controls (NC), n=6; age 85-88 SP Stage 0-A) were used for [3H]MK801 (0.017μCi/cc) autoradiographic studies.

Results FDGluMK801 was synthesized in approx. 13% yield and was lower than previously reported fluorodeoxyglucamine derivatives (Baranwal et al., 2015). [18F]FDGluMK801 was prepared in low radiochemical yields (<10%) determined using chromatographic methods. Binding affinity of FDGluMK801 was found to be in the micromolar range, compared to the nanomolar affinity measured for parent MK-801. Binding of [3H]MK801 in AD FC was 1.7 times higher than NC and addition of glutamate showed a significantly greater effect in AD FC compared to NC. These results indicate a significant increase in glutamate-induced 3H-MK801 binding in AD brains.

Conclusions Our preliminary results of FDGluMK801 show promise as a possible imaging and therapeutic agent, as it has a similar binding affinity to the NMDA receptor ion-channel compared to one of the currently used therapeutic drugs, Memantine (IC50 ~0.50 µM). Since in vivo binding of NMDA ion-channel blockers may be irreversible, the moderate affinity and the amphiphilic nature of [18F]FDGluMK801 may enable visualization of the ion-channel using PET. Further studies are underway to evaluate [18F]FDGluMK801and confirm our preliminary results which indicate that NMDA receptor activity is increased significantly in AD brains in the presence of glutamate compared to normal controls. Research Support: NIH AG 029479