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Efficacy of the ketamine metabolite (2R,6R)-hydroxynorketamine in mice models of pain
  1. Jeffrey S Kroin,
  2. Vaskar Das,
  3. Mario Moric and
  4. Asokumar Buvanendran
  1. Department of Anesthesiology, Rush University Medical Center, Chicago, Illinois, USA
  1. Correspondence to Asokumar Buvanendran, Department of Anesthesiology, Rush University Medical Center, Chicago, IL 60612, USA; asokumar{at}aol.com

Abstract

Background and objectives Ketamine has been shown to reduce chronic pain; however, the adverse events associated with ketamine makes it challenging for use outside of the perioperative setting. The ketamine metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) has a therapeutic effect in mice models of depression, with minimal side effects. The objective of this study is to determine if (2R,6R)-HNK has efficacy in both acute and chronic mouse pain models.

Methods Mice were tested in three pain models: nerve-injury neuropathic pain, tibia fracture complex regional pain syndrome type-1 (CRPS1) pain, and plantar incision postoperative pain. Once mechanical allodynia had developed, systemic (2R,6R)-HNK or ketamine was administered as a bolus injection and compared with saline control in relieving allodynia.

Results In all three models, 10 mg/kg ketamine failed to produce sustained analgesia. In the neuropathic pain model, a single intraperitoneal injection of 10 mg/kg (2R,6R)-HNK elevated von Frey thresholds over a time period of 1–24hours compared with saline (F=121.6, p<0.0001), and three daily (2R,6R)-HNK injections elevated von Frey thresholds for 3 days compared with saline (F=33.4, p=0.0002). In the CRPS1 model, three (2R,6R)-HNK injections elevated von Frey thresholds for 3 days and then an additional 4 days compared with saline (F=116.1, p<0.0001). In the postoperative pain model, three (2R,6R)-HNK injections elevated von Frey thresholds for 3 days and then an additional 5 days compared with saline (F=60.6, p<0.0001).

Conclusions This study demonstrates that (2R,6R)-HNK is superior to ketamine in reducing mechanical allodynia in acute and chronic pain models and suggests it may be a new non-opioid drug for future therapeutic studies.

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Introduction

Ketamine infusions have been used since the 1960s to treat acute pain, but that usage has recently exploded, becoming a mainstay of treatment in the perioperative period among individuals with refractory pain and among opioid-tolerant patients.1 Over the past two decades, the use of intravenous ketamine infusions as a treatment for chronic pain has increased dramatically, with wide variation in patient selection, dosing and monitoring. Short-term intravenous infusions of ketamine have been shown to reduce chronic pain for days or weeks afterwards, although not all studies show efficacy.2 However, the adverse events (eg, dissociate side effects) profile of ketamine, and the potential for abuse, makes it difficult to use long term in the chronic pain population even at subanesthetic doses.2 In a recent clinical study of oral ketamine administration for postoperative pain, ketamine doses were limited to 1.0 mg/kg, due to hallucinations at higher doses.3

After systemic injection, ketamine is stereoselectively metabolized via P450 enzymatic transformations into a broad array of metabolites.4 5 Following intravenous ketamine administration in humans, the total plasma exposure of its metabolite (2S,6S;2R,6R)-hydroxynorketamine is higher than that of the parent drug.6 A study in mice has demonstrated that a single intraperitoneal injection of the ketamine metabolite enantiomer (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) has a therapeutic effect in mice models of depression.4 Moreover, (2R,6R)-HNK does not have the anesthetic or sedative effects of ketamine itself even at very high doses in mice, and at subanesthetic doses it does not have abuse/addiction liability.4 There are no published studies on the effect of injections of (2R,6R)-HNK in chronic or postoperative pain models. However, there is considerable overlap between chronic pain and depression in terms of both co-prevalence and treatment, with many therapies typically used to treat one being effective for the other, including ketamine.2 A single intraperitoneal injection of ketamine itself has a short-term therapeutic effect in a mice model of complex regional pain syndrome type-1 (CRPS1),7 and clinical infusions of ketamine have been used to treat complex regional pain syndrome.8

The central hypothesis of this study is to demonstrate that injection of (2R,6R)-HNK has efficacy in three mouse pain models: nerved-injury neuropathic pain, CRPS1 pain, and postoperative pain. Although opioids are widely used in clinical practice to treat chronic pain, the effectiveness of opioids for chronic pain has been questioned.9 In addition, the recent surge in opioid use and overdoses has led to a rise in non-opioid-based treatment options. The importance in pursuing ketamine and its metabolites for pain management is to both to improve the patient’s quality of life and to reduce the nation’s dependence on opioids.

Methods

Drugs and doses

(2R,6R)-HNK (Sigma-Aldrich, St. Louis, Missouri, USA) was dissolved in normal saline,4 at 1.33 mg/mL. When administered intraperitoneally in 0.15 mL volume, the dose is 0.2 mg, which in a 0.02 kg mouse is 10 mg/kg. In comparison experiments, the parent compound ketamine ((±)-ketamine hydrochloride, Sigma-Aldrich) was also dissolved in normal saline, at 1.33 mg/mL, and administered intraperitoneally in 0.15 mL volume for a 10 mg/kg dose. The 10 mg/kg intraperitoneal dose of (2R,6R)-HNK and ketamine were chosen for this pain study because that dose had a potent and sustained antidepressant effect in several mouse models of depression.4

Neuropathic pain model

The Spared Nerve Injury (SNI) model in mice, in which the left peroneal and sural nerves are cut, but the tibial nerve is spared, produces mechanical allodynia in the hindpaw within a few days that lasts for months.10 Female young adult 20 g CD-1 mice (Charles River, Wilmington, Maine, USA) underwent left SNI surgery,10 under 1.5% isoflurane anesthesia.

Prior to surgery and 7 days after SNI surgery, mice were tested for ipsilateral von Frey force withdrawal thresholds. In all of our experiments, von Frey testing was conducted mid-day (noon to 15:00). When von Frey filaments are applied to the mid-plantar surface of the left (ipsilateral) hind paw, if there is decrease in the force withdrawal threshold at 7 days, then that is interpreted as mechanical allodynia.11 Typically, normal mice have 2.5–4 g thresholds and neuropathic mice<1 g. At day 7, the mice were then allocated into two groups having virtually identical low von Frey thresholds: half of the mice receive intraperitoneal drug and half of the mice receive intraperitoneal saline.

Single injection experiments

Mice were administered a single 10 mg/kg intraperitoneal injection of (2R,6R)-HNK (n=6) or saline (n=6) and retested for ipsilateral von Frey thresholds at 1 hour, 8 hours, 24 hours, 48 hours, and 72 hours after injection. In a new group of mice after SNI, the same single injection experiment was performed with 10 mg/kg intraperitoneal ketamine (n=6) or saline (n=6).

Multiple injection experiment

If any of the drugs in the single injection experiment showed an antiallodynic effect, we then investigated the efficacy of three daily injections at 10 mg/kg of drug (n=6) or saline (n=6) in SNI mice. Mice were retested for ipsilateral von Frey thresholds at 24 hours after each injection (just prior to the next injection) and until there were no longer differences between drug and saline groups.

CRPS-1 chronic pain model

Female young adult 20 g C57BL/6J mice (Jackson Labs, Bar Harbour, Maine, USA) underwent a closed distal tibia facture in the right leg, under 1.5% isoflurane anesthesia.7 12 A 0.011″ stainless steel pin was inserted percutaneously through the entire length of the tibia to promote good bone union.13 After injury, the hindlimb was wrapped using casting tape so the hip, knee and ankle were all fixed. At the end of 3 weeks, casts were removed.7 12

Prior to surgery and 28 days after tibia fracture, mice were tested for ipsilateral von Frey force withdrawal thresholds, applied to the mid-plantar surface of the right hind paw. Then the mice were allocated into two groups, having virtually identical low von Frey thresholds: half of the mice received daily intraperitoneal injections of 10 mg/kg (2R,6R)-HNK (n=7) and half of the mice received intraperitoneal injections of saline (n=7), for three consecutive days. Mice were retested for ipsilateral von Frey thresholds at 24 hours after each injection (just prior to the next injection) and until there were no longer differences between drug and saline groups. As a comparison, after a 12-day washout period, 10 mg/kg ketamine versus saline was also injected on three consecutive days (with the drug and saline groups reversed from the (2R,6R)-HNK experiment).

Postoperative pain model

Female young adult 20 g CD-1 mice (Charles River) underwent a left plantar hindpaw incision, under 1.5% isoflurane anaesthesia. Briefly, under aseptic conditions, a 5 mm long incision was made in the plantar hindpaw and the underlying muscle elevated with a fine forceps. The skin was then closed with a 7–0 mattress suture. This has been shown to produce mechanical hyperalgesia lasting 4–5 days in mice.14

Prior to surgery and 4 hours after plantar incision, mice were tested for ipsilateral von Frey force withdrawal thresholds, applied to the mid-plantar surface of the left hind paw. Typically, normal mice have 2.5–4 g thresholds and 4 hours after plantar incision <1 g.14 After 4 hours, the mice were then allocated into two groups having virtually identical low von Frey thresholds: half of the mice received daily intraperitoneal injections of 10 mg/kg (2R,6R)-HNK (n=7) and half of the mice received intraperitoneal injections of saline (n=7), for three consecutive days. Mice were retested for ipsilateral von Frey thresholds at 24 hours after each injection (just prior to the next injection) and until there were no longer differences between drug and saline groups. In a separate experiment, at 4 hours after plantar incision, the mice were allocated into two groups having virtually identical low von Frey thresholds: half of the mice received daily intraperitoneal injections of 10 mg/kg ketamine (n=7) and half of the mice received intraperitoneal injections of saline (n=7), for three consecutive days. Mice were retested for ipsilateral von Frey thresholds at 24 hours after each injection (just prior to the next injection) and until there were no longer differences between drug and saline groups.

The SNI model and the tibia fracture CRPS model do not exhibit thermal hyperalgesia,10 15 however, the plantar incision model does,14 and so a new experiment was performed to measure the response to a radiant heat stimulus in plantar hindpaw incision mice with three daily injections of (2R,6R)-HNK versus saline. Prior to surgery and 4 hours after plantar incision, mice were tested for ipsilateral paw withdrawal latencies, with the radiant bulb heat focused on the mid-plantar surface of the left hind paw (Paw Thermal Stimulator System, Department of Anesthesiology, University of California, San Diego, USA).16 Mice were tested on a glass platform within a plastic enclosure. The bulb current was set for a 12 s paw withdrawal latency in normal mice, and 4 hours after plantar incision the latency is typically<4 s.14 After 4 hours, the mice were then allocated into two groups having virtually identical low withdrawal latencies: half of the mice received dailyintraperitoneal injections of 10 mg/kg (2R,6R)-HNK (n=7) and half of the mice received intraperitoneal injections of saline (n=7), for three consecutive days. Mice were retested for ipsilateral paw withdrawal latencies at 24 hours after each injection (just prior to the next injection) and until there were no longer differences between drug and saline groups. At each time point, the result of two trials 15 min apart was averaged to provide the average paw withdrawal latency.

Postoperative pain model: naloxone test

Since the underlying motive for studying (2R,6R)-HNK is to find a non-opioid drug for postoperative and chronic pain, the plantar foot incision von Frey experiment was repeated to provide evidence that (2R,6R)-HNK does not have morphine-like opioid properties. After obtaining presurgery von Frey withdrawal thresholds, a plantar foot incision was performed. After 4 hours, the mice were then allocated into two groups having virtually identical low von Frey thresholds: half of the mice received daily injections of subcutaneous saline (0.05 mL) first, and then 10 min later, intraperitoneal 10 mg/kg (2R,6R)-HNK (n=7) and half of the mice received subcutaneaous 1 mg/kg naloxone (naloxone hydrochloride injection, Akorn, Lake Forest, Illinois, USA) first, and then 10 min later, intraperitoneal 10 mg/kg (2R,6R)-HNK (n=7), for three consecutive days. The subcutaneous 1 mg/kg naloxone dose, 15 min before 10 mg/kg morphine, provided full blockade of the analgesic effect of morphine in the foot-shock test in rats,17 and a 1 mg/kg naloxone dose, 10 min before morphine, blocked morphine-induced analgesia in the tail-flick test in mice.18 Mice were retested for ipsilateral von Frey thresholds at 24 hours after each injection (just prior to the next injection) and for at least 5 days after the last injection.

Statistical analysis

Sample size was designed for comparing von Frey thresholds in mice pain models after (2R,6R)-HNK versus saline injections. Based on the Zanos et al’s study of (2R,6R)-HNK injection in models related to depression, a minimum of n=6/group was estimated for each experiment to see an effect of the compound.4 Von Frey force thresholds or paw withdrawal latencies over time were compared between groups using repeated measures mixed model with autoregressive lag 1 covariance structure, with preplanned contrasts. Error inflation due to multiple testing (different time points) was controlled using the Holm-Bonferroni method. Model-based assumptions were evaluated and adjusted if necessary and threshold for significance was set at 0.05. All analyses were performed using SAS V.9.4.

Results

Neuropathic pain model

(2R,6R)-HNK experiment, single injection

Prior to nerve injury, there was no difference in von Frey thresholds between the (2R,6R)-HNK and saline groups (figure 1A). At 7 days after SNI surgery, ipsilateral von Frey thresholds were greatly reduced from presurgery levels (p<0.0001, for the (2R,6R)-HNK group or the saline group). A single intraperitoneal injection of 10 mg/kg (2R,6R)-HNK in SNI mice elevated ipsilateral von Frey thresholds over a time period of 1–24 hours compared with saline (F=121.6, p<0.0001). This group difference was seen at all time points: 1 hour (p<0.0001), 4 hours (p<0.0001), and 24 hours (p<0.0001). By 48 hours after drug injection, there was no longer a difference between groups.

Figure 1

Spared nerve injury (SNI) mice with ipsilateral mechanical allodynia (decreased von Frey force withdrawal thresholds). Antihypersensitivity effects of 10 mg/kg intraperitoneal drugs versus saline. (A) (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), single injection reduced allodynia (increased thresholds) for 24 hours. (B) Ketamine, single injection did not reduce allodynia. (C) (2R,6R)-HNK, three daily injections reduced allodynia for 3 days. ****Drug different from saline, p<0.0001; n=6/group for drug and saline mice. Data are presented as mean and SE. preSNI, mice before nerve injury; SNI BL, mice with neuropathic pain.

Ketamine experiment, single injection

Prior to nerve injury, there was no difference in von Frey thresholds between the ketamine and saline group (figure 1B). At 7 days after SNI surgery, ipsilateral von Frey thresholds were greatly reduced from presurgery levels (p<0.0001, for the ketamine group or the saline group). In contrast to (2R,6R)-HNK, a single intraperitoneal injection of 10 mg/kg ketamine in SNI mice did not affect ipsilateral von Frey thresholds over a time period of 1–24 hours compared with saline (F=0.111, p=0.7464).

(2R,6R)-HNK experiment, multiple injection

Since a single injection of (2R,6R)-HNK increased von Frey thresholds for 24 hours, we wanted to see what the effect would be of daily injections of that compound. Prior to nerve injury, there was no difference in ipsilateral von Frey thresholds between the (2R,6R)-HNK and saline groups (figure 1C). After SNI surgery, ipsilateral von Frey thresholds were greatly reduced from presurgery levels (p<0.0001, for the (2R,6R)-HNK group or the saline group). Multiple 10 mg/kg (2R,6R)-HNK injections (daily for 3 days) elevated von Frey thresholds (at 24 hours after each injection) for 3 days compared with saline (F=33.4, p=0.0002). This group difference was seen at all time points: 1 day (p<0.0001), 2 days (p<0.0001), and 3 days (p<0.0001). However, there was no ‘memory’ effect by which von Frey thresholds were still elevated at 2 days (48 hours) after the last injection. In our study, no animals experienced adverse events from (2R,6R)-HNK.

CRPS type-1 chronic pain model

(2R,6R)-HNK experiment, multiple injection

Prior to distal tibia fracture, there was no difference in von Frey thresholds between the (2R,6R)-HNK and saline groups (figure 2A). After fracture, ipsilateral von Frey thresholds were greatly reduced from preinjury levels (p<0.0001, for the (2R,6R)-HNK group or the saline group). Multiple 10 mg/kg (2R,6R)-HNK injections (daily for 3 days) elevated ipsilateral von Frey thresholds (at 24 hours after each injection) for 3 days, and then for up to 4 days after the third injection, compared with saline (F=116.1, p<0.0001). This group difference was seen at all time points: 1 day (p<0.0001), 2 days (p<0.0001), 3 days (p=0.0001) and at 4 days after the third injection (p<0.0001). So, unlike the SNI neuropathic pain model, there was a ‘memory’ effect by which von Frey thresholds were still elevated 4 days after the last injection.

Figure 2

Complex regional pain syndrome (CRPS) mice with ipsilateral mechanical allodynia. Antihypersensitivity effects of 10 mg/kg intraperitoneal drugs versus saline. (A) (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), three daily injections reduced allodynia (increased thresholds) for 3 days, plus four more days after third injection. (B) Ketamine, three daily injections did not reduce allodynia. ****Drug different from saline, p<0.0001; n=7/group for drug and saline mice. Data are presented as mean and SE. Prefracture, mice before tibia fracture/repair; CRPS BL, mice with postfracture pain.

Ketamine experiment, multiple injection

Prior to distal tibia fracture, there was no difference in von Frey thresholds between the ketamine and saline groups (figure 2B). After fracture, ipsilateral von Frey thresholds were greatly reduced from preinjury levels (p<0.0001, for the (2R,6R)-HNK group or the saline group). Multiple 10 mg/kg ketamine injections (daily for 3 days) did not elevate ipsilateral von Frey thresholds over the 3 days (F=1.79, p=0.2081).

Postoperative pain model

(2R,6R)-HNK experiment, multiple injection

Prior to plantar hindpaw incision, there was no difference in von Frey thresholds between the (2R,6R)-HNK and saline groups (figure 3A). At 4 hours after incision, ipsilateral von Frey thresholds were greatly reduced from preincision levels (p<0.0001, for the (2R,6R)-HNK group or the saline group). Multiple 10 mg/kg (2R,6R)-HNK injections (daily for 3 days) elevated ipsilateral von Frey thresholds (at 24 hours after each injection) for 3 days, and then for up to 5 days after the third injection, compared with saline (F=60.6, p<0.0001). This group difference was seen at all time points: 1 day (p<0.0001), 2 day (p<0.0001), 3 day (p<0.0001), 2 days after the third injection (p<0.0001), and at 5 days after the third injection (p<0.0001). So, again, unlike the SNI model, there was a ‘memory’ effect by which von Frey thresholds were still elevated 5 days after the last injection.

Figure 3

Plantar foot incision postoperative pain mice with ipsilateral hypersensitivity. Antihypersensitivity effects of 10 mg/kg intraperitoneal drugs versus saline. (A) (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), three daily injections reduced mechanical allodynia (increased thresholds) for 3 days, plus five more days after third injection. ****Drug different from saline, p<0.0001; n=7/group for drug and saline mice. (B) Ketamine, three daily injections did not reduce mechanical allodynia. n=7/group for drug and saline mice. (C) (2R,6R)-HNK, three daily injections reduced thermal hyperalgesia (increased paw withdrawal latencies) for 3 days, plus five more days after third injection. ****Drug different from saline, p<0.0001; ***drug different from saline, p<0.001; **drug different from saline, p<0.01; n=7/group for drug and saline mice. (D) Naloxone preinjection did not block the antiallodynic effect of (2R,6R)-HNK. n=7/group for naloxone and saline mice. Data are presented as mean and SE. Preincision, mice before hindpaw incision; 4-hour postincision, mice with postoperative pain.

Ketamine experiment, multiple injection

Prior to plantar hindpaw incision, there was no difference in von Frey thresholds between the ketamine and saline groups (figure 3B). At 4 hours after plantar hindpaw incision, ipsilateral von Frey thresholds were greatly reduced from preincision levels (p<0.0001, for the (2R,6R)-HNK group or the saline group). Multiple 10 mg/kg ketamine injections (daily for 3 days) did not elevate ipsilateral von Frey thresholds over the 3 days (F=0.031, p=0.8632).

(2R,6R)-HNK Experiment, multiple injection, thermal hyperalgesia

Prior to plantar hindpaw incision, there was no difference in ipsilateral paw withdrawal latencies between the (2R,6R)-HNK and saline groups (figure 3C). At 4 hours after incision, ipsilateral paw withdrawal latencies were greatly reduced from pre-incision levels (p<0.0001, for the (2R,6R)-HNK group or the saline group). Multiple 10 mg/kg (2R,6R)-HNK injections (daily for 3 days) elevated ipsilateral paw withdrawal latencies (at 24 hours after each injection) for 3 days and then for up to 5 days after the third injection, compared with saline (F=59.3, p<0.0001). This group difference was seen at all time points: 1 day (p=0.0016), 2 days (p=0.0001), 3 days (p<0.0001), 2 days after the third injection (p<0.0001), and at 5 days after the third injection (p=0.0044). Therefore, the antihyperalgesic effect of (2R,6R)-HNK was very similar for both thermal and von Frey stimuli.

(2R,6R)-HNK Experiment, multiple injection, Naloxone Test

Prior to plantar hindpaw incision, there was no difference in von Frey thresholds between the naloxone and saline groups (figure 3D). At 4 hours after incision, ipsilateral von Frey thresholds were greatly reduced from preincision levels (p<0.0001, for the naloxone and saline groups). There were no differences in ipsilateral von Frey thresholds (at 24 hours after each injection) over three injection days and then for up to 5 days after the third injection between the naloxone and saline groups (F=0.03, p=0.8706), both of which also received 10 mg/kg (2R,6R)-HNK injections. Over the three injection days and up to 5 days after, both the saline group (p=0.0003) and the naloxone group (p=0.0002) had elevated ipsilateral von Frey thresholds (antiallodynic effect) compared with the 4-hour postincision time point.

Discussion

Multiple intraperitoneal injections of the ketamine metabolite enantiomer (2R,6R)-HNK were able to reduce mechanical allodynia for days in two chronic pain models and one postoperative pain model. The parent compound ketamine did not produce sustained analgesia (ie, lasting for 24 hours after an injection) in any of these pain models using our daily bolus injection protocol.

In the SNI neuropathic pain model, daily 10 mg/kg (2R,6R)-HNK for 3 days maintained elevated von Frey force withdrawal thresholds for 3 days, while daily 10 mg/kg ketamine did not increase von Frey thresholds. In a recent paper, with a different nerve injury neuropathic pain mouse model, 15 mg/kg ketamine twice a day for 10 days did elevate von Frey thresholds after about 6 days of injection, and the effect lasted for days after injections ceased.19 Therefore, with greater and more prolonged dosing, we may have also seen some efficacy with ketamine in the SNI model, but at the lower dose and duration that we used in the present study (2R,6R)-HNK was the more effective compound.

In a mouse tibia fracture CRPS1 model, there was a persistent antiallodynic effect that lasted 4 days beyond the last injection of (2R,6R)-HNK. This is not a pharmacokinetic effect, since the half-life of (2R,6R)-HNK in the mouse brain is less than 1 hour.4 This suggests that (2R,6R)-HNK may reduce the central sensitization that can contribute to CRPS1 pain.20 Although with our daily intraperitoneal boluses of ketamine, 10 mg/kg for 3 days, we did not see any elevation of von Frey thresholds, in a similar study with the CRPS1 mouse tibia fracture model, slow subcutaneous infusion of ketamine at 2 mg/kg/day for 7 days starting at 7 weeks postfracture (ours was at 6 weeks postfracture) did produce sustained elevation of von Frey thresholds.12 So, ketamine infusion does have effectiveness in CRPS1 mice, but in our bolus protocol (2R,6R)-HNK was the more potent compound. A recent clinical review of intravenous ketamine infusion in chronic pain patients showed some efficacy, but higher dosages and more frequent infusions were associated with greater risks of adverse events.2 That is why there is a need for an alternative to ketamine itself.

In the plantar hindpaw incision postoperative pain model, there was also a persistent antiallodynic effect that lasted 5 days beyond the last injection of (2R,6R)-HNK, but no antiallodynic effect of ketamine any time point. (2R,6R)-HNK also produced a persistent antihyperalgesic effect to noxious thermal stimulation that lasted 5 days beyond the last injection in the plantar hindpaw incision model, so the compound shows efficacy in two sensory modalities. Interestingly, in the direct nerve injury SNI neuropathic pain model, which could be considered a mouse CRPS-2 model, there was no prolongation of the antiallodynic effect beyond 24 hours after the last drug injection. So in terms of any sustained effect of (2R,6R)-HNK in reducing mechanical allodynia, the CRPS1 fracture model seems to have more in common with the postoperative pain model than with the neuropathic pain model. This may be because both the CRPS1 model and the postoperative pain model produce intense peripheral inflammation, while the nerve injury model is a milder nerve-regeneration inflammation. In preclinical models, inflammatory hyperalgesia is frequently sensitive to agents such as non-steroidal anti-inflammatory drugs whereas neuropathic pain states are not.21

Projecting to clinical usage, (2R,6R)-HNK is not associated with the many side effects of ketamine. At a dose of 125 mg/kg, an order of magnitude higher than we used in our study, (2R,6R)-HNK did not produce motor incoordination (rotarod test) in mice over the first hour after injection.4 In contrast, ketamine at 10 mg/kg, the dose we used in our study, caused motor incoordination within 10 min after injection.4 In addition, unlike ketamine, (2R,6R)-HNK had low abuse/addiction liability, tested with a drug self-administration model in mice. Zanos et al concluded that ‘overall (2R,6R)-HNK administration revealed an innocuous side effect profile compared with ketamine’.4

The main limitation of our study is that the drug doses and the dose timing (bolus injection) were adopted from a paper on the efficacy of (2R,6R)-HNK in treating depression in mice. The reason for using that dosing was that it took advantage of the long duration of action of (2R,6R)-HNK (in the depression model) and the low side effect profile of (2R,6R)-HNK compared with the parent compound ketamine.4 Therefore, for each of the three pain models used in this study, we could have increased the (2R,6R)-HNK and ketamine doses above 10 mg/kg, or used more frequent dosing, or extended the duration of drug injections beyond 3 days. In addition, we could have used multiple measures of pain testing, each set best suited to each pain model. However, mechanical allodynia was one measure that could easily be compared among all three pain models.

In conclusion, ketamine has been used for the treatment of chronic pain and depression, and there are common factors that indicate neurophysiological overlap between the two diseases.2 So it is not that surprising that the efficacy of (2R,6R)-HNK in a mouse depression model would carry over to mouse pain models. One possible link between the two disorders is the hippocampus,22 which is involved with pain processing and is altered during depression. Glutamate and its receptor subtypes, N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), have been implicated in the pathogenesis of both depression and chronic pain.23 At present, there is some controversy about the primary site of action of (2R,6R)-HNK in depression models. Zanos et al gave behavioural and pharmacological evidence that (2R,6R)-HNK, unlike ketamine, was not acting at the NMDA glutamate receptor, but most likely at the AMPA glutamate receptor.4 However, another study in cultured hippocampal neurons showed that (2R,6R)-HNK inhibited synaptic NMDA receptors.24 Since our naloxone study demonstrated in the plantar hindpaw incision model that (2R,6R)-HNK does not act at the mu-opioid receptor, the combination of prolonged analgesia with low side effects makes (2R,6R)-HNK an attractive candidate for pain control.

References

Footnotes

  • Funding This work was supported by University Anesthesiologists, S.C., Chicago, Illinois, USA.

  • Competing interests None declared.

  • Ethics approval IACUC of Rush University Medical Center.

  • Provenance and peer review Not commissioned; externally peer reviewed

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