A Pharmacological Evaluation of the Analgesic Effect and Hippocampal Protein Modulation of the Ketamine Metabolite (2R,6R)-Hydroxynorketamine in Murine Pain Models

Abstract

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 (AUC 0-3d ) was calculated using trapezoidal integration. The AUC 0-3d 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 AUC 0-3d 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.

Figure 1:

Box plots and dot plots with box plots of the effect of pretreatment with naloxone and NBQX on the antiallodynic efficacy of (2R,6R)-HNK in murine models of pain. The solid line is the median, the dashed line is the mean, the box represents the 25^th to 75^th percentiles, the whiskers the 10^th and 90^th percentiles and the solid circle the 5^th and 95^th percentiles. Upper Panel: Plantar incision model. A: Box plot of paw withdrawal thresholds prior to surgery, prior to drug administration and 24 hours following drug administration for 3 consecutive days. B: Dot and box plot of AUC_0–3d among treatment groups. † = NBQX + (2R,6R)-HNK different from saline + (2R,6R)-HNK, difference −3.64 g*d (95% CI −5.08 to −2.21, P<0.001) and NBQX + (2R,6R)-HNK different from naloxone + (2R,6R)-HNK, difference −3.68 g*d (95% CI −6.60 to −0.72, P<0.001). Data analyzed using Dunn’s test. Middle panel: Spared neve injury. A: Box plot of paw withdrawal thresholds prior to surgery, prior to drug administration and 24 hours following drug administration for 3 consecutive days. A: Box plot of paw withdrawal thresholds prior to surgery, prior to drug administration and 24 hours following drug administration for 3 consecutive days. B: Dot and box plot of AUC_0–3d among treatment groups. † = NBQX + (2R,6R)-HNK different from saline + (2R,6R)-HNK, difference −6.09 g*d (95% CI −7.83 to −4.35, P<0.001) and NBQX + (2R,6R)-HNK different from naloxone + (2R,6R)-HNK, difference −5.15 g*d (95% CI −7.98 to −2.30, P<0.001). Data analyzed using Dunn’s test. Lower Panel: Tibial fracture model. A: Box plot of paw withdrawal thresholds prior to surgery, prior to drug administration and 24 hours following drug administration for 3 consecutive days. B: Dot and box plot of AUC_0–3d among treatment groups. † = NBQX + (2R,6R)-HNK different from saline + (2R,6R)-HNK, difference −5.18 g*d (95% CI −7.58 to −2.78, P<0.001) and NBQX + (2R,6R)-HNK different from naloxone + (2R,6R)-HNK, difference −4.95 g*d (95% CI −8.01 to −1.95, P<0.001). Data analyzed using Dunn’s test.

Figure 2:

Dot plots with box plots of protein/GAPDH ratios for saline and (2R,6R)-HNK treated naive mice. The solid line is the median, the dashed line is the mean, the box represents the 25^th to 75^th percentiles, the whiskers the 10^th and 90^th percentiles and the solid circle the 5^th and 95^th percentiles. † = (2R,6R)-HNK different from saline treated animals, P<0.05. Sex adjusted mean differences in GluA1 0.081 (95% CI 0.006 to 0.157, P=0.035), GluA2 0.046 (95% CI −0.023 to 0.11, P=0.19), pKv2.1 0.037 (95% CI −0.015 to 0.088, P=0.17), pCaMKII 0.072 (−0.031 to 0.175, P=0.17), and BDNF −0.080 (95% CI −0.111 to −0.047, P<0.001). Differences not adjusted for multiple comparisons. Representative immunoblots from the right hippocampus of 3 mice treated with saline and 3 treated with (2R,6R)-HNK. Blots of protein of interest and GAPDH (housekeeping protein) are taken from the same lane on the chromatograph.

Figure 3:

Dot plots with box plots of protein/GAPDH ratios for saline and (2R,6R)-HNK treated animals following PI surgery. The solid line is the median, the dashed line is the mean, the box represents the 25^th to 75^th percentiles, the whiskers the 10^th and 90^th percentiles and the solid circle the 5^th and 95^th percentiles. † = (2R,6R)-HNK different from saline vehicle animals, P<0.05. Sex adjusted mean difference in GluA1 0.187 (95% CI 0.111 to 0.263, P<0.001), GluA2 0.154 (95% CI 0.031 to 0.276, P=0.014), pKv2.1 0.196 (95% CI 0.115 to 0.276, P<0.001), pCaMKII 0.363 (95% CI 0.208 to 0.517, P<0.001), and BDNF −0.094 (95% CI −0.171 to −0.016, P<0.017). Differences not adjusted for multiple comparisons. Representative immunoblots from the right hippocampus of 3 mice treated with saline and 3 treated with (2R,6R)-HNK. Blots of protein of interest and GAPDH (housekeeping protein) are taken from the same lane on the chromatograph.

Figure 4:

Dot plots with box plots of protein/GAPDH ratios for saline and (2R,6R)-HNK treated animals following SNI surgery. The solid line is the median, the dashed line is the mean, the box represents the 25^th to 75^th percentiles, the whiskers the 10^th and 90^th percentiles and the solid circle the 5^th and 95^th percentiles. † = (2R,6R)-HNK different from saline vehicle animals, P<0.05. Adjusted mean difference in GluA1 0.070 (95% CI −0.055 to 0.196, P<0.393), GluA2 0.226 (95% CI 0.129 to 0.322, P<0.001), pKv2.1 0.246 (95% CI 0.155 to 0.336, P<0.001), pCaMKII 0.274 (95% CI 0.199 to 0.348, P<0.001), and BDNF −0.224 (95% CI −0.302 to −0.147, P<0.001). Differences not adjusted for multiple comparisons. Representative immunoblots from the right hippocampus of 3 mice treated with saline and 3 treated with (2R,6R)-HNK. Blots of protein of interest and GAPDH (housekeeping protein) are taken from the same lane on the chromatograph.

Figure 5:

Dot plots with box plots of protein/GAPDH ratios for saline and (2R,6R)-HNK treated animals following TF surgery. The solid line is the median, the dashed line is the mean, the box represents the 25^th to 75^th percentiles, the whiskers the 10^th and 90^th percentiles and the solid circle the 5^th and 95^th percentiles. † = (2R,6R)-HNK different from saline vehicle animals, P<0.05. Adjusted mean differences and 95% confidence intervals for GluA1 0.131 (95% CI 0.073 to 0.189, P<0.001), GluA2 0.203 (95% CI 0.120 to 0.285, P<0.001), pKv2.1 0.196 (95% CI 0.120 0.273, P<0.001), pCaMKII 0.180 (95% CI 0.106 to 0.253, P<0.001), and BDNF −0.208 (95% CI −0.304 to −0.113, P<0.001). Differences not adjusted for multiple comparisons. Representative immunoblots from the right hippocampus of 3 mice treated with saline and 3 treated with (2R,6R)-HNK. Blots of protein of interest and GAPDH (housekeeping protein) are taken from the same lane on the chromatograph.