Background & Aims
The N-methyl-D-aspartate receptor (NMDAr) has the ability to provide significant analgesic relief without the side effects associated with opioid use. Previous studies have shown that agmatine, a decarboxylated form of L-arginine, is antihyperalgesic in pre-clinical models of neuropathic pain. This antihyperalgesic effect is believed to be mediated through antagonism of the GluN2B subunit of NMDArs located in the dorsal horn of the spinal cord. Agmatine’s development as a therapeutic has been hindered by poor oral bioavailability and blood brain barrier permeability. We developed a strategically substituted analog of agmatine (SSA3) that displays improved biopharmaceutical characteristics relative to the parent compound, agmatine. While SSA3 has demonstrated antihyperalgesic efficacy in vivo, the molecular mechanism of this analog has yet to be determined. We characterized the subunit selectivity, conversion to agmatine, and efficacy in the tail flick assay of hyperalgesia.
Methods
SSA3 antagonism of spinal NMDArs was measured as a decrease in the excitatory postsynaptic current (EPSC) amplitude and duration from neurons in lamina II of the dorsal horn. Nav1.8-ChR2-expressing mice were used for this experiment to selectively activate Nav1.8-expressing nociceptive afferents. 470nm blue light was shone through a 60x objective and EPSCs were recorded. Increasing concentrations of NMDA inhibitors were administered and responses from each concentration were averaged. For the incubation experiment, hemisected spinal cords were incubated in aCSF with or without 10mM SSA3 for 10 minutes. The tissue was removed, processed, and analyzed with LC-MS to determine agmatine concentration. For tail flick, Mice were intrathecally injected with NMDA to induce transient thermal hypersensitivity, which was assessed via warm water tail immersion. SSA3 was intrathecally injected prior to i.t. NMDA. Latency to tail flick was measured and plotted relative to baseline
Results
Similar to the results seen in Waataja, Peterson et al. 2019, agmatine concentration-dependently inhibited NMDAr-mediated currents selectively at GluN2B-containing NMDArs. SSA3 was also able to concentration-dependently inhibit NMDAr-mediated EPSCs, also apparently at GluN2B-containing NMDArs. The latency to peak and duration observed in the difference currents for the highest concentration of SSA3 is similar to that of other GluN2B-selective antagonists used in this study. When hemisected spinal cord tissue is incubated in SSA3, we observed a dramatic increase in agmatine concentration, relative to the untreated section of spinal cord. In tail flick thermal hyperalgesia, we observed an increase in withdrawal latency, shown as a minimal decrease in tail withdrawal latency relative to baseline, when SSA3 is administered (i.t.) 10 minutes prior to i.t. NMDA.
Conclusions
The electrophysiological experiments performed here suggest that SSA3 is acting at NMDArs in spinal cord slices in a manner pharmacodynamically similar to that of agmatine. The agmatine level in the spinal cord showed a trend of elevation following 10 min SSA3 incubation, implying SSA3 is being metabolized to agmatine during the 10 min incubation with spinal cord tissues. Our tail flick data suggest that intrathecally administered SSA3 is able to inhibit postsynaptic nNOS, which is believed to be the primary enzyme responsible for the thermal hyperalgesia observed following intrathecal NMDA administration.
References
Presenting Author
Lukas Caye
Poster Authors
Lukas Caye
University of Minnesota
Lead Author
Christina Peterson
University of Minnesota
Lead Author
Kelley Kitto
University of Minnesota, Dept. of Pharmaceutics
Lead Author
Tongzhen Xie
University of Minnesota
Lead Author
Carolyn Fairbanks
PhD
University of Minnesota, Dept. of Pharmaceutics
Lead Author