A Pharmacological Evaluation of the Analgesic Effect and Hippocampal Protein Modulation of the Ketamine Metabolite (2R,6R)-Hydroxynorketamine in Murine Pain Models
Authors: Das Vaskar , Basovich Michael B. , Thomas Craig J. , Kroin Jeffrey S. , Buvanendran Asokumar , McCarthy Robert J. 
Published: June 2023

BACKGROUND:
The ketamine metabolite (2R,6R)-hydroxynorketamine ([2R,6R]-HNK) has analgesic efficacy in murine models of acute, neuropathic, and chronic pain. The purpose of this study was to evaluate the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) dependence of (2R,6R)-HNK analgesia and protein changes in the hippocampus in murine pain models administered (2R,6R)-HNK or saline.

METHODS:
All mice were CD-1 IGS outbred mice. Male and female mice underwent plantar incision (PI) (n = 60), spared nerve injury (SNI) (n = 64), or tibial fracture (TF) (n = 40) surgery on the left hind limb. Mechanical allodynia was assessed using calibrated von Frey filaments. Mice were randomized to receive saline, naloxone, or the brain-penetrating AMPA blocker (1,2,3,4-Tetrahydro-6-nitro-2,3-dioxobenzo [f]quinoxaline-7-sulfonamide [NBQX]) before (2R,6R)-HNK 10 mg/kg, and this was repeated for 3 consecutive days. The area under the paw withdrawal threshold by time curve for days 0 to 3 (AUC0-3d) was calculated using trapezoidal integration. The AUC0-3d was converted to percent antiallodynic effect using the baseline and pretreatment values as 0% and 100%. In separate experiments, a single dose of (2R,6R)-HNK 10 mg/kg or saline was administered to naive mice (n = 20) and 2 doses to PI (n = 40), SNI injury (n = 40), or TF (n = 40) mice. Naive mice were tested for ambulation, rearing, and motor strength. Immunoblot studies of the right hippocampal tissue were performed to evaluate the ratios of glutamate ionotropic receptor (AMPA) type subunit 1 (GluA1), glutamate ionotropic receptor (AMPA) type subunit 2 (GluA2), phosphorylated voltage-gated potassium channel 2.1 (p-Kv2.1), phosphorylated-calcium/calmodulin-dependent protein kinase II (p-CaMKII), brain-derived neurotrophic factor (BDNF), phosphorylated protein kinase B (p-AKT), phosphorylated extracellular signal-regulated kinase (p-ERK), CXC chemokine receptor 4 (CXCR4), phosphorylated eukaryotic translation initiation factor 2 subunit 1 (p-EIF2SI), and phosphorylated eukaryotic translation initiation factor 4E (p-EIF4E) to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

RESULTS:
No model-specific gender difference in antiallodynic responses before (2R,6R)-HNK administration was observed. The antiallodynic AUC0-3d of (2R,6R)-HNK was decreased by NBQX but not with pretreatment with naloxone or saline. The adjusted mean (95% confidence interval [CI]) antiallodynic effect of (2R,6R)-HNK in the PI, SNI, and TF models was 40.7% (34.1%–47.3%), 55.1% (48.7%–61.5%), and 54.7% (46.5%–63.0%), greater in the SNI, difference 14.3% (95% CI, 3.1–25.6; P = .007) and TF, difference 13.9% (95% CI, 1.9–26.0; P = .019) compared to the PI model. No effect of (2R,6R)-HNK on ambulation, rearing, or motor coordination was observed. Administration of (2R,6R)-HNK was associated with increased GluA1, GluA2, p-Kv2.1, and p-CaMKII and decreased BDNF ratios in the hippocampus, with model-specific variations in proteins involved in other pain pathways.

CONCLUSIONS:
(2R,6R)-HNK analgesia is AMPA-dependent, and (2R,6R)-HNK affected glutamate, potassium, calcium, and BDNF pathways in the hippocampus. At 10 mg/kg, (2R,6R)-HNK demonstrated a greater antiallodynic effect in models of chronic compared with acute pain. Protein analysis in the hippocampus suggests that AMPA-dependent alterations in BDNF-TrkB and Kv2.1 pathways may be involved in the antiallodynic effect of (2R,6R)-HNK.