Behavioural and pharmacological effects of cannabidiol (CBD) and the cannabidiol analogue KLS-13019 in mouse models of pain and reinforcement

Abstract

Background and Purpose

Cannabidiol (CBD) is a non-euphorigenic component of Cannabis sativa that prevents the development of paclitaxel-induced mechanical sensitivity in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). We recently reported that the CBD structural analogue KLS-13019 shows efficacy in an in vitro model of CIPN. The present study was to characterize the behavioural effects of KLS-13019 compared to CBD and morphine in mouse models of CIPN, nociceptive pain and reinforcement.

Experimental Approach

Prevention or reversal of paclitaxel-induced mechanical sensitivity were assessed following intraperitoneal or oral administration of CBD, KLS-13019 or morphine. Antinociceptive activity using acetic acid-induced stretching and hot plate assay, anti-reinforcing effects on palatable food or morphine self-administration and binding to human opioid receptors were also determined.

Key Results

Like CBD, KLS-13019 prevented the development of mechanical sensitivity associated with paclitaxel administration. In contrast to CBD, KLS-13019 was also effective at reversing established mechanical sensitivity. KLS-13019 significantly attenuated acetic acid-induced stretching and produced modest effects in the hot plate assay. KLS-13019 was devoid of activity at μ-, δ- or κ-opioid receptors. Lastly, KLS-13019, but not CBD, attenuated the reinforcing effects of palatable food or morphine.

Conclusions and Implications

KLS-13019 like CBD, prevented the development of CIPN, while KLS-13019 uniquely attenuated established CIPN. Because KLS-13019 binds to fewer biological targets, this will help to identifying molecular mechanisms shared by these two compounds and those unique to KLS-13019. Lastly, KLS-13019 may possess the ability to attenuate reinforced behaviour, an effect not observed in the present study with CBD.

Abbreviations

CBD

cannabidiol

CIPN

chemotherapy-induced peripheral neuropathy

mNCX-1

mitochondrial sodium/calcium exchanger 1

What is already known

• The non-euphorigenic phytocannabinoid cannabidiol prevents mechanical sensitivity in mice treated with the chemotherapeutic agent paclitaxel.
• CBD analogue KLS-13019 shows efficacy in an in vitro model of chemotherapy-induced peripheral neuropathy (CIPN).

What does this study add

• KLS-13019 also prevented mechanical sensitivity to paclitaxel following intraperitoneal and oral administration in mice.
• KLS-13019 reversed established mechanical sensitivity and attenuated morphine self-administration in mice, while CBD was ineffective.

What is the clinical significance

• CBD is currently in clinical trials for chemotherapy-induced peripheral neuropathy as well as opioid relapse.
• KLS-13019 has the potential to be a more potent, effective, orally bioavailable option to CBD.

1 INTRODUCTION

Neuropathic pain is a pathophysiologic condition produced by damage to, or pathological changes in, the peripheral nervous system and CNS. It is characterized by abnormal pain sensations, including spontaneous pain, hyperalgesia (i.e. increased sensitivity to a typically noxious stimulus) and allodynia (i.e. increased sensitivity to a typically non-noxious stimulus) that lack an apparent physiological function. Neuropathic pain remains a challenging neurologic disorder that adversely affects quality of life and presents a large unmet medical need for improved therapies. Chemotherapy-induced peripheral neuropathy (CIPN) is a progressive, enduring and often irreversible condition featuring pain, numbness, tingling and sensitivity to cold in the hands and feet (sometimes progressing to the arms and legs) that afflicts between 30% and 40% of patients undergoing chemotherapy (Gutierrez-Gutierrez et al., 2010). Chemotherapy drugs associated with CIPN include the vinca alkaloids vincristine and vinblastine, the taxanes paclitaxel and docetaxel, the proteasome inhibitors such as bortezomib and the platinum-based drugs cisplatin, oxaliplatin and carboplatin. Current treatments for neuropathic pain are few and of limited effectiveness. The three major classes of compounds most often used are tricyclic antidepressants (TCAs), anticonvulsants (AEDs) and, to a lesser extent, μ-opioid receptor agonists. μ-agonists, such as morphine, are not recommended for chronic use in neuropathic pain patients due to lack of evidence of long-term benefit coupled with increased risk of harm (Chou et al., 2015). In a recent report, however, upwards of 97% of CIPN patients reported using prescription opioids for pain management even though there is only weak evidence that long-term continuation of opioids provides clinically significant pain relief in these patients (Shah et al., 2018). Now more than ever, this is of critical importance. Of the 20.5 million Americans 12 or older that had a substance use disorder in 2015, 2 million had a substance use disorder involving prescription pain relievers. Sales of prescription pain relievers in 2010 were four times those in 1999 and the substance use disorder treatment admission rate in 2009 was six times the 1999 rate (http://www.asam.org/docs/default-source/advocacy/opioid-addiction-disease-facts-figures.pdf). Therefore, there is a clear and pressing need for novel treatment strategies to decrease or replace opioid analgesic use for treatment of neuropathic pain.

Recently, there has been resurgence in interest in the potential medical utility of the Cannabis plant and its constituents, and mechanism-based basic research is warranted to develop safe and effective cannabinoid-based pain treatments. Cannabidiol (CBD) is a non-psychoactive component of Cannabis sativa that is neuroprotective, independent of cannabinoid receptors (Hampson et al., 1998). We have recently reported that CBD prevents the development of paclitaxel-induced mechanical sensitivity in mice in vivo (King et al., 2017; Ward et al., 2011, 2014). Based on this preclinical work, CBD is also currently being tested in two separate clinical trials for the treatment of CIPN in cancer patients. Interestingly, CBD has also been shown to decrease opioid craving both in a rat model (Ren et al., 2009) and in heroin users (Hurd et al., 2019). This has important implications for the possibility of CBD-based treatment approaches for neuropathic pain and mitigation of opioid use disorder.

CBD has limitations in terms of oral bioavailability and its relative potency and safety continue to be under investigation. KannaLife Sciences has generated a structural analogue of CBD, KLS-13019, that shows improved bioavailability as compared with CBD (Kinney et al., 2016). Moreover, KLS-13019 was more potent than CBD in reducing paclitaxel-induced neurotoxicity in vitro (Brenneman et al., 2018). KLS-13019 also possesses significantly fewer molecular interactions as compared with CBD, lacking appreciable binding affinity for CB₂, 5-HT_1A, TRPV1 and PPAR-γ (NR1C3) receptors (Brenneman et al., 2018). This poses an opportunity for a safer, more effective compound that will also increases our knowledge pertaining to the relevant mechanisms of action of CBD and like molecules for the treatment of CIPN and other conditions. Most recently, we demonstrated the importance of mitochondrial Ca²⁺ regulation in the actions of both CBD and KLS-13019 in preventing neurotoxicity associated with paclitaxel administration. Specifically, we determined that the mitochondrial sodium/calcium exchanger 1 (mNCX-1) was involved in the neuroprotective actions of both CBD and KLS-13019 in vitro (Brenneman et al., 2019).

The goal of the present study was to characterize the behavioural pharmacological effects of KLS-13019 and compare them to CBD and morphine in male mice, with a focus on prevention and reversal of paclitaxel-induced mechanical sensitivity. Previously, we also determined that CBD was ineffective at attenuating thermal nociception using the hot plate assay but was fully effective at preventing visceral pain in the acetic acid-induced stretching assay (Neelakantan et al., 2015), so we also determined the effectiveness of KLS-13019 in these assays. We determined the effect of CBD or KLS-13019 on palatable food or morphine reinforcement. Lastly, we tested both CBD and KLS-13019 for binding affinity for μ-, δ- and κ-opioid receptors using a radioligand binding assay.

2 METHODS

Studies were designed to generate groups of equal size, using randomization and blinded analysis, except where noted. All group sizes are n = 5/12 per group, and exact group numbers are specified in the figure legends. These group sizes are based on several years of experience and published work with these mouse models by the authors. All animal care and experimental procedures complied with the guidelines of the Temple University Institutional Animal Care and Use Committee and were as humane as possible. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, 2015; Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020), as well as the US NIH guidelines for reporting experiments involving animals.

2.1 Animals

Male C57Bl/6 (CIPN, reinforcement studies) and Swiss Webster (acetic acid-induced stretching, hot plate) mice were purchased from Taconic Farms (Cranbury NJ, USA) weighing 18–20 g for C57's and 20–25 g for Swiss Webster's upon arrival. C57Bl/6 mice have historically been used in our laboratory for all CIPN experiments based on an initial report by Smith et al. (2004) determining relative sensitivity to paclitaxel-induced nociceptive behaviours across mouse strains and follow-up work in house. At the end of all experiments mice were killed by CO₂ asphyxiation followed by cervical dislocation. Mice were not reused.

Swiss Webster mice were used for hot plate and acetic acid tests because of their relative sensitivities to morphine antinociception in the hot plate and acetic acid as compared with C57's. Mice were group housed in plastic cages and allowed to acclimate to the temperature-controlled and humidity-controlled animal facility for 5 days before the experiments began. Mice had free access to food and water except where noted in the operant behaviour assay procedures (see below).

2.2 Chemotherapy-induced peripheral neuropathy (CIPN) prevention

2.2.1 Experiment 1

After acclimatization in the vivarium, mechanical allodynia was assessed using von Frey monofilaments of varying forces (0.07–4.0 g) applied to the mid-plantar surface of the right hind paw, with each application held in c-shape for 6 s. A modified up-down method as described in Ward et al. (2011) was used. Mice were placed in individual Plexiglas compartments (Med Associates, St. Albans, VT, USA) on top of a wire grid floor suspended 20 cm above the laboratory bench top and acclimatized to the environment for 30 min before each test session. Baseline sensitivity to the monofilaments was assessed 1–3 days before the start of drug administration and again on Experimental Day 14. On Experimental Days 1, 3, 5 and 7, mice received the following two i.p. injections, spaced 15 min apart:- first injection vehicle, morphine (10 mg·kg^-1), CBD (2.5 mg·kg^-1) or KLS-13019 (2.5 mg·kg^-1); second injection saline or paclitaxel (8.0 mg·kg^-1). The 15 min pretreatment time for CBD, KLS-13019 and morphine was based on our previous work with CBD and its protective effects in this assay.

2.2.2 Experiment 2

The effect of oral administration was tested in a separate cohort of mice. All experimental details were identical to above, except that the first treatments on Days 1, 3, 5 and 7 were administered per os (p.o.): CBD (0.25, 2.5 or 25 mg·kg^-1) and KLS-13019 (0.25, 2.5 or 25 mg·kg^-1).

Doses were selected based on previous work with CBD and morphine in this model and previously reported pharmacokinetic data with KLS-13019. All mechanical allodynia testing was done by an experimenter blinded to the treatment conditions.

2.3 Chemotherapy-induced peripheral neuropathy (CIPN) reversal

2.3.1 Experiment 3

The ability of these compounds to reverse established CIPN was tested in a separate cohort of mice. After acclimatization in the vivarium, mechanical allodynia was assessed as described above. Baseline sensitivity to the monofilaments was assessed 1–3 days before the start of drug administration. Saline or paclitaxel was administered i.p. on Days 1, 3, 5 and 7. Mechanical sensitivity was again assessed on Day 11 to demonstrate mechanical sensitivity in the paclitaxel-treated group. Vehicle, morphine (20 mg·kg^-1), CBD (20 mg·kg^-1) or KLS-13019 (20 mg·kg^-1) were administered i.p. on Days 12, 13 and 14. Mechanical allodynia was reassessed on Day 14, 15 min following the last injection. Doses and 3-day dosing regimen were based on previous preliminary experiments with CBD and morphine and a published report with gabapentin (Xiao et al., 2007), where both morphine and gabapentin, but not CBD, were shown to reverse established mechanical allodynia under this dosing protocol.

2.3.2 Experiment 4

In a separate cohort of mice, additional doses of KLS-13019 were tested i.p. in a within-subjects design, starting with the 1.25 mg·kg^-1 dose on Days 12, 13 and 14; the 2.5 mg·kg^-1 dose on Days 19, 20 and 21; and the 5.0 mg·kg^-1doses on Days 26, 27 and 28. Mechanical sensitivity in a saline alone group, paclitaxel alone group and the KLS-13019 + paclitaxel group was tested on Days 14, 21 and 28. Dosing was not randomized so that the effect of 1.25 mg·kg^-1 could be determined, and either a lower or higher dose could be followed up with depending upon the results.

2.3.3 Experiment 5

The effect of oral administration was tested in a separate cohort of mice. All experimental details were identical to that described for Experiment 3, except that KLS-13019 (0.25, 2.5 or 25 mg·kg^-1) was administered p.o. on Days 12, 13 and 14 in a between-subjects design.

All mechanical allodynia testing was done by an experimenter blinded to the treatment conditions.

2.4 Acetic acid-induced stretching

One hundred per cent of glacial acetic acid was diluted to a concentration of 0.4% with physiological saline (0.9% sodium chloride). The pH of the solution was then checked to ensure proper acidity (pH 2.7) prior to any experimentation and made fresh each day.

2.4.1 Experiment 6

Mice were brought to the testing room, individually placed in fresh housing chambers and allowed to acclimate for 15 min. Mice were treated i.p. with morphine (0.1 and 1.0 mg·kg^-1), CBD (5.6 and 56 mg·kg^-1) or KLS-13019 (5.6 and 56 mg·kg^-1) and then returned to these cages. These doses were taken from a previous study for our laboratory (Neelakantan et al., 2015). Thirty minutes later, mice were then treated i.p. with saline or 0.4% acetic acid and returned to the test chambers. After 2 min elapsed, the mice were recorded on video for 20 min using an FHD high-definition camcorder. Upon completion, the videos were then scored for acetic acid-induced stretching behaviour by an experimenter blinded to the treatment conditions.

2.5 Hot plate

2.5.1 Experiment 7

The effects of morphine, CBD or KLS-13019 (1.0–100 mg·kg^-1; i.p^.) were assessed using a hot plate thermal nociceptive assay with the temperature and experimental parameters based on our previous studies using morphine and CBD in C57Bl/6 mice (Fischer et al., 2010; Neelakantan et al., 2015). In this procedure, a single mouse was placed on the hot plate set to a temperature of 56°C with a 15-cm high plastic cylinder. The latency to when the mouse licked its hind paw, jumped off the hot plate or a cut-off time of 25 s was recorded. Two control latencies spaced 5 min apart were determined, and the mean value for each individual mouse was taken as the baseline latency measure. Mice were then administered morphine, CBD or KLS-13019 at 1.0 mg·kg^-1and tested on the hot plate 30 min later to determine post-drug latencies. Immediately following the first test, mice were administered 2.0 mg·kg^-1of the same drug to test the 3.0 mg·kg^-1 doses for a cumulative dosing procedure and were tested on the hot plate 30 min later. This cumulative dosing procedure continued until all doses were tested for a drug for each mouse.

2.6 Operant studies

2.6.1 Apparatus

Palatable food and morphine self-administration experiments were conducted in standard mouse operant experimental chambers (21.6 cm × 17.8 cm × 12.7 cm, Model ENV-307W, Med Associates, Georgia, VT, USA). The experimental chambers were located within ventilated sound-attenuating enclosures and each chamber was equipped with the following: two nose-poke holes (1.2-cm diameter), one on the left and one on the right, with internal amber stimulus lights (ENV-313W), a centre dipper hole between the two nose-pokes that opened to a motor-driven dipper (ENV-302W) for liquid food presentation, a house light (ENV-315M), a tone generator (ENV-323AW) and a ventilator fan. The receptacle for dipper access contained an amber stimulus light located above (ENV-221M). In addition, an electronic circuit operated a computer-controlled syringe pump designed for intravenous drug delivery. The syringe was connected to a single-channel fluid swivel mounted on a counter-balanced arm above the operant chamber (MED-307A-CT-B2).

2.7 Procedure

2.7.1 Training

All mice we initially trained to make operant responses to obtain access to a 50% solution of the liquid nutritional drink Ensure® diluted with tap water. Mice were food-restricted to maintain 90% of their baseline body weight and trained to respond for liquid Ensure® in the right nose-poke under a fixed ratio (FR1) schedule of reinforcement during daily 1-h sessions. During each session, every response in the right illuminated nose-poke hole resulted in illumination of the stimulus light and delivery of liquid food through the centre dipper receptacle for 20 s. Responses on inactive nose-poke had no scheduled consequences during the experimental sessions. The criteria for acquisition of liquid food self-administration were defined as three consecutive days of stable fixed ratio 1 responding (<10% changes among the mean number of reinforcers earned for the 3 days) and at least 75% of total responses corresponding to the active right nose-pokes.

2.7.2 Experiment 8

Mice that met criteria for fixed ratio 1 (FO1) acquisition were then changed to a progressive ratio (PR) schedule of reinforcement. Under this schedule, the session length was increased to 4 h and the response requirements to earn a single reinforcer were increased exponentially in the following progression: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62 and so forth (Richardson & Roberts, 1996). The dependent variable measured was the final ratio requirement completed by the end of the 4 h. The corresponding number of reinforcers earned was designated as the breakpoint. The criteria for stable response under the progressive ratio schedule were defined as three consecutive days of stable breakpoints (<20% changes from the mean number of reinforcers earned for three consecutive days). Once each mouse met these criteria, the following injection schedule was introduced: three consecutive days of vehicle injections and then three consecutive days of cannabinoid injection. This was repeated until all doses of CBD and KLS-13019 were assessed in each mouse. The order of testing was CBD from the highest to lowest dose, followed by KLS-13019 from the lowest to highest dose.

2.8 Morphine self-administration

2.8.1 Experiment 9

A separate group of mice were trained to respond for liquid Ensure® under an fixed ratio 1 schedule and then trained to self-administer morphine. Once mice met stable criteria for liquid food, they were surgically implanted with a chronic indwelling jugular cannula as described in Ward et al. (2011) and Caine et al. (1999). Following surgery and 2 days of recovery, mice were trained to self-administer 0.1 and 0.3 mg·kg^-1 per infusion morphine under an fixed ratio 1 schedule of reinforcement during daily 2-h sessions, because the optimal training dose for morphine differed across subjects. These doses of morphine were chosen based on our previous work with morphine self-administration in C57Bl/6 mice (Neelakantan et al., 2016). During the session, every response on the active right nose-poke resulted in a single infusion of morphine paired with illumination of the light above the food receptacle and a tone delivery for 1 s. After every morphine infusion, the chamber houselights went off for 60 s and the mouse's responses had no programmed consequences. After stable responding under fixed ratio schedule (<10% changes among the mean number of reinforcers earned for 3 days and with at least 75% of total responses corresponding to the active right nose-pokes), mice were given access to morphine under an fixed ratio 3 schedule with 0.1 mg·kg^-1 per infusion morphine for three additional days and then to 0.1 mg·kg^-1 per infusion morphine under the same progressive ratio schedule of reinforcement described above. Once each mouse met the progressive ratio criteria, the following injection schedule was introduced: - three consecutive days of vehicle injections, three consecutive days of KLS-13019 (2.5 mg·kg^-1) injection, three consecutive days of vehicle injection and then three consecutive days of CBD (2.5 mg·kg^-1) injection.

2.9 Radioligand opioid binding studies

CBD and KLS-13019 were screened with human opiate receptors in recombinant HEK-293 cells (CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045) for their responses in radioligand binding assays. The goal of these studies was to explore the possibility that KLS-13019 may interact with opiate receptors to mediate the reversal effect on neuropathic pain. All of the opiate receptor binding screens were conducted at a single concentration of 10 μM in competition binding assays in which the test compounds were added concurrently with the radiolabelled ligand. The choice of 10 μM as a test concentration was utilized to capture both low affinity interactions and to coincide with the concentration of CBD that produced full efficacy neuroprotection from paclitaxel toxicity in dorsal root ganglion cultures.

These studies were conducted by Eurofins Panlabs Discovery Services (New Taipei City, Taiwan). CBD and KLS-13019 were screened in duplicate assays at 10 μM in a vehicle of 1% DMSO. All assays were conducted in an incubation buffer consisting of 50-mM Tris–HCl, pH 7.4. Opiate δ (OP1, DOP) receptor antagonist binding studies were conducted with 1.3-nM [³H]-naltrindole in 60-min incubations at 25°C. Non-specific binding was determined in the presence of 1-μM naltrindole. The reference compound was naltrindole (Kd 1.8 nM). Opiate κ (OP2, KOP) antagonist binding studies were conducted with 0.60-nM [³H]-diprenorphine in 60-min incubations at 25°C. Non-specific binding was determined in the presence of 10-μM naloxone. The reference compound was U-69593 (Kd 29 nM). Opiate κ (OP2, KOP) agonist binding studies were conducted with 1-nM 3H-U-69593 in 90-min incubations at 25°C. Non-specific binding was determined in the presence of 10-μM U-69593. The reference compound was U-69593 (Kd 1.3 nM). Opiate μ (OP3, MOP) receptor antagonist binding assays were conducted with 0.6-nM [³H]-diprenorphine in 60-min incubations at 25°C. Non-specific binding was determined in the presence of 10-μM naloxone. The reference compound was DAMGO (Kd 9 nM). Opiate μ (OP3, MOP) receptor agonist binding assays were conducted with 3-nM [³H]-morphine in 60-min incubations at 25°C. Non-specific binding was determined in the presence of 10-μM diprenorphine. The reference compound was DAMGO (Kd 1.2 nM). Nociceptin opioid peptide (NOP/ORL-1) receptor binding assays were conducted with 0.6-nM [³H]-nociceptin in 60-min incubations at 25°C. Non-specific binding was determined in the presence of 1-μM of the ligand nociceptin/orphanin-FQ (N/OFQ). The reference compound was orphanin-FQ (Kd 0.6 nM; no significant inhibition at any of the human opiate receptors). Eurofins Panlabs criteria for significance in this assay are set at >50% binding inhibition.

2.10 Materials

The synthesis of KLS-13019 has been described previously in detail (Kinney et al., 2016). Verification of the structural identity for KLS-13019 was determined by ¹H NMR, LC/UV and LC/MS. The purity of KLS-13019 was 98.6% as determined by LC/MS. CBD and morphine sulfate were obtained through the National Institute on Drug Abuse (NIDA) Drug Supply Program (Bethesda, MD, USA). The purity of CBD was certified to be >99% pure and was confirmed by a third-party certificate of analysis as well. CBD and KLS-13019 were dissolved in a vehicle of 1:1:18 ethyl alcohol:cremophor:saline (v/v). Ethyl alcohol, Cremophor EL®, and 9% sodium chloride solution were purchased from Millipore Sigma (St. Louis, MO, USA). Morphine was dissolved in 0.9% saline. Paclitaxel was obtained as a 6 mg·ml^-1 concentration stock solution (Hospira, Inc., Lake Forest, IL, USA) and then further diluted in saline. As CBD is a natural product extracted from Cannabis sativa, these studies are reported in compliance with the recommendations made by the British Journal of Pharmacology (Izzo et al., 2020).

2.11 Data and analyses

The design and analysis of experiments presented in this manuscript comply with the recommendations of the British Journal of Pharmacology and requirements on experimental design and analysis (Curtis et al., 2018). The declared group sizes are the number of independent values, and the statistical analyses were done using these independent values. No data exclusion criteria were set, and therefore, no outliers were excluded from the data analysis or presentation. All sample sizes are listed for each experimental groups in the corresponding figure legends, where n = number of independent values. One-way or two-way ANOVAs and Dunnett's post-hoc comparisons (GraphPad Prism 8, RRID:SCR_002798) were used to determine doses significantly different from vehicle treatment. Post hoc tests were only conducted if F values achieved statistical significance as defined as P < .05.

2.12 Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos, et al., 2019; Alexander, Mathie, et al., 2019).

3 RESULTS

3.1 CIPN prevention

3.1.1 Experiment 1

The 2.5 mg·kg^-1 doses of CBD and KLS-13019 both prevented the development of mechanical sensitivity, while prophylactic treatment with 10 mg·kg^-1 morphine failed to impact its development. One-way ANOVA demonstrates a significant effect of treatment, post-hoc test analysis shows that CBD and KLS-13019 pretreatment significantly decreased mechanical sensitivity as compared with the vehicle-treated paclitaxel group (Figure 1).

FIGURE 1

Effects of morphine, CBD or KLS-13019 on prevention of paclitaxel-induced mechanical sensitivity. Morphine, CBD or KLS-13019 was administered IP 15 min prior to injection of paclitaxel on Days 1, 3, 5 and 7. Mechanical sensitivity was measured using von Frey filaments on Day 0 and again on Day 14. N = 7 per Veh + Veh group, 8 per all other treatment groups. * indicates significantly different from paclitaxel alone group, P < .05. Data plotted as mean ± SEM

3.1.2 Experiment 2

Oral administration of CBD prevented development of mechanical sensitivity. One-way ANOVA showed a main effect of treatment with Dunnett's post-hoc test showing the 25 mg·kg^-1 dose as significant. Oral administration of KLS-13019 also prevented development of mechanical sensitivity. One-way ANOVA showed a main effect of treatment with Dunnett's Dunnett's post-hoc test showing the 2.5 and 25 mg·kg^-1 doses as significant (Figure 2).

FIGURE 2

Effect of orally administered CBD or KLS-13019 on prevention of paclitaxel-induced mechanical sensitivity. CBD (left panel) or KLS-13019 (right panel) was administered orally 15 min prior to injection of paclitaxel on Days 1, 3, 5 and 7. Mechanical sensitivity was measured using von Frey filaments on Day 0 and again on Day 14. For CBD, n = 8 per Sal, 0 and 25 mg·kg^-1 groups, N = 7 per 0.25 and 2 mg·kg^-1 treatment groups. For KLS, n = 8 per Sal, 0, 0.25 and 25 mg·kg^-1 treatment groups, n = 6 per 2.5 mg·kg^-1 treatment group. * indicates significantly different from paclitaxel alone group, P < .05. Data plotted as mean ± SEM

3.2 CIPN reversal

3.2.1 Experiments 3–5

There was a significant effect of i.p. treatment on the reversal of established CIPN, with Dunnett's post-hoc test revealing significance with morphine and KLS-13019 versus vehicle in paclitaxel-treated mice. CBD treatment did not reverse established CIPN (Figure 3 left panel). After further testing of a wider range of KLS-13019 doses, two-way ANOVA showed a main significant effect of treatment, but not dose, or interaction. Dunnett's post-hoc test showed a significant difference between KLS-13019 treatment and paclitaxel alone at the 2.5 and 5.0 mg·kg^-1 doses (Figure 3 middle panel). Oral administration of KLS-13019 also significantly reversed established CIPN. Dunnett's post-hoc test revealed a significant effect at the 2.5 mg·kg^-1 dose (Figure 3 right panel).

FIGURE 3

Effect of CBD or KLS-13019 on reversal of an established paclitaxel-induced mechanical sensitivity. Saline or paclitaxel (PAC) was administered on Days 1, 3, 5 and 7, and treatment with morphine, CBD or KLS-13019 started on Day 12. Left panel: Starting on Day 12, mice were treated for 3 days with morphine, CBD or KLS-13019 i.p. in the morning, and mechanical sensitivity was tested in the afternoon of Day 14. Middle panel: Starting on Day 11, mice were treated for 3 days with one of three doses of KLS-13019 i.p., followed by a washout period of 4 days and then repeated with a new dose of KLS-13019 to test a total of three doses in each mouse. Right panel: Starting on Day 12, mice were treated for 3 days with KLS-13019 PO in the morning, and mechanical sensitivity was tested in the afternoon of Day 14. n = 8 per group for left and middle panels, n = 5 per group for right panel. * indicates significantly different from paclitaxel alone group P < .05. Data plotted as mean ± SEM

3.3 Acetic acid-induced stretching

3.3.1 Experiment 6

Treatment with morphine, CBD or KLS-13019 significantly reduced acetic acid-induced stretching. Dunnett's post-hoc test revealed a significant effect of all treatments at all doses. The highest tested dose of morphine, CBD or KLS-13019 did not induce stretching behaviour alone (Figures 4).

FIGURE 4

Effect of morphine, CBD or KLS-13019 on acetic acid-induced stretching behaviour. Mice were treated i.p. with morphine, CBD or KLS-13019 followed 30 min later with an i.p. injection of saline or 0.4% acetic acid. n = 8 per group. * indicates significantly different from acetic acid alone group, P < .05. Data plotted as mean ± SEM

3.4 Hot plate

3.4.1 Experiment 7

Morphine produced significant dose-dependent antinociception on the 56°C hot plate. Dunnett's post-hoc test shows significant increase in latency at the 10, 30 and 100 mg·kg^-1 doses. As we reported previously, CBD produced marginal efficacy on the hot plate. Dunnett's post-hoc test shows significant increase in latency at the 100 mg·kg^-1 dose. KLS-13019 produced dose-dependent antinociception on the hot plate. Dunnett's post-hoc test shows significant increase in latency at all doses tested (Figure 5).

FIGURE 5

Effect of morphine, CBD or KLS-13019 on thermal antinociception as measured on the hot plate. Dose–response curves for paw withdrawal latency for morphine, CBD and KLS-13019 were generated using a cumulative dosing procedure on the 56°C hot plate with a 25-s cut-off. n = 8 per group. * indicates significantly different from acetic acid alone group, P < .05. Data plotted as mean ± SEM

3.5 Operant studies

3.5.1 Experiment 8

KLS-13019 significantly attenuated motivation to respond for liquid Ensure®, as determined by one-way ANOVA. Dunnett's post-hoc test revealed a significant effect with the 5.0 and 20 mg·kg^-1 doses. By comparison, CBD treatment did not affect progressive ratio responding for liquid food (Figure 6 left panel).

FIGURE 6

Effect of CBD or KLS-13019 on food- or morphine-reinforced behaviour. Mice were trained to respond for liquid 50% vanilla-flavoured ensure under a fixed ratio (FR) schedule of reinforcement. Mice were then switched to a progressive ratio (PR) schedule of reinforcement for Ensure (left panel) or to an FR and then PR schedule of reinforcement for morphine (0.1 mg·kg^-1 per infusion). Saline, CBD or KLS-13019 was administered i.p. 15 min prior to the PR self-administration session. The final ratio completed under the PR schedule is plotted along the Y axis. n = 12 for food maintained responding. For morphine self-administration, n = 12 for saline, 9 for KLS and 8 for CBD. * indicates P < .05 as compared with saline control, P < .05. Data plotted as mean ± SEM

3.5.2 Experiment 9

KLS-13019, but not CBD, also attenuated morphine self-administration maintained by a progressive ratio schedule of reinforcement. One-way ANOVA revealed a main effect of treatment with Dunnett's post-hoc test showing a significant difference between the saline- and KLS-13019-treated groups (Figure 6 right panel).

3.6 Opioid binding studies

Responses in binding inhibition were concluded to be significant if ≥50%. By this a priori standard, CBD exhibited significant inhibition at all human opioid receptors and KLS-13019 produced significant inhibition at none of the human opioid receptors (Figure 7).

FIGURE 7

Comparison of CBD and KLS-13019 on human opioid receptor binding. CBD and KLS-13019 were screened in radioligand binding assays with human opiate receptors in recombinant HEK-293 cells. All opiate receptor binding screens were conducted at a single concentration of 10 μM in competition binding assays in which the test compounds were added concurrently with the radiolabelled ligand. All assays were conducted in an incubation buffer consisting of 50-mM Tris–HCl, pH 7.4. Each value is the mean of duplicate determinations. Eurofins Panlabs criteria for significance in this assay are set at >50% binding inhibition

4 DISCUSSION

The overarching goal of the experiments reported here was to determine the behavioural effects of KLS-13019, a structural analogue of CBD. Specifically, we aimed to determine whether KLS-13019 was as potent and effective as CBD at mitigating mechanical allodynia associated with paclitaxel treatment and whether it was efficacious following oral administration. Overall, our results demonstrate that KLS-13019 prevents the development of paclitaxel-induced mechanical sensitivity in male C57Bl/6 mice similar to CBD and was effective when administered either i.p. or orally. As expected, these compounds were effective in a prophylactic model in contrast to the gold standard analgesic morphine, which was without protective effects. Results following oral administration suggest that oral KLS-13019 may be more potent than oral CBD. In the present study, oral KLS-13019 was effective at the 2.5 mg·kg^_1 dose, whereas CBD showed statistically significant effects at 25 mg·kg^-1, an effect less potent than we previously reported for CBD following intraperitoneal administration, where the observed minimal effective dose was 1.0 mg·kg^-1 (King et al., 2017). These results are in line with our previous pharmacokinetic data showing improved bioavailability of KLS-13019 as compared with CBD following oral administration (Kinney et al., 2016).

An even more notable difference was observed in the efficacy of CBD versus KLS-13019 in their ability to reverse an already established mechanical sensitivity. The analgesic effects of morphine significantly decreased established paclitaxel-induced mechanical sensitivity. CBD, which has been shown to lack antinociceptive effects in physiological nociception models (Neelakantan et al., 2015; Silva et al., 2017; Sofia et al., 1975), did not reverse mechanical sensitivity in the model, replicating previous unpublished data from our laboratory across a wider range of CBD doses (5.0–20 mg·kg^-1). In the present study, KLS-13019 reversed established CIPN at a minimal effective dose of 2.5 mg·kg^-1 i.p. and p.o. These results indicate that in addition to the neuroprotective mechanisms of CBD and KLS-13019, such as regulation of mitochondrial Ca²⁺ handling (Brenneman et al., 2019), KLS-13019 may work through an additional, unique mechanism. Based on the present opioid receptor binding data for KLS-13019 showing no appreciable binding at μ-, δ-, or κ-opioid receptors at 10 μM, it is unlikely that these reversal effects observed were opioid receptor mediated. We are currently exploring mechanisms unique to KLS-13019 that explain its distinctive ability to reverse established paclitaxel-induced mechanical sensitivity as compared with CBD. It should also be noted that the highest dose of CBD tested in this assay was 20 mg·kg^-1, a dose more than 10X higher than that needed to prevent CIPN. Given the complex dose–response relationships observed by us and others (e.g. King et al., 2017), it is possible that higher doses of CBD may be effective in the reversal paradigm and need to be explored.

Based on the ability of KLS-13019 to both prevent and reverse paclitaxel-induced mechanical sensitivity, we proceeded to test KLS-13019 in comparison with morphine and CBD in two additional rodent models predictive of antinociceptive drugs, acetic acid-induced stretching and hot plate. We and others have previously reported that CBD treatment attenuates stretching behaviour induced by intraperitoneal injection of acetic acid (Neelakantan et al., 2016; Silva et al., 2017). Here, we demonstrate that like CBD and morphine, KLS-13019 significantly attenuates this stretching behaviour. Acetic acid induces an inflammatory response in the abdominal cavity, with subsequent activation of nociceptors (Collier et al., 1968). Acute inflammation emerges in the peritoneal area, and stretching behaviour induced by acetic acid is a nonselective antinociceptive model, as acetic acid acts indirectly by inducing the release of several endogenous mediators that stimulate visceral nociceptive neurons. Acetic acid-induced stretching can be attenuated by anti-inflammatory agents and opioid analgesics, as well as other centrally active drugs (Vaz et al., 1996). The mechanism by which CBD attenuates acetic acid-induced stretching has yet to be determined but is independent of cannabinoid CB₁ or CB₂ receptors (Silva et al., 2017). We and others have reported that CBD attenuates the release of a wide range of pro-inflammatory mediators in other rodent models of pain (Britch et al., 2020; Li et al., 2018; Linher-Melville et al., 2020). It remains to be determined whether KLS-13019 is working through CBD-like anti-inflammatory mechanisms in this model or through a unique pathway similar to its effects on the reversal of paclitaxel-induced mechanical sensitivity.

Based on the reversal effects of KLS-13019, we also determined whether KLS-13019 would attenuate thermal nociception as measured by the hot plate. We and others have previously reported that CBD produces little to no antinociceptive effects in this assay (Neelakantan et al., 2016; Sofia et al., 1975) at doses up to 90 mg·kg^-1 (Silva et al., 2017). In the present study, CBD produced modest but significant increase in paw withdrawal latency at the 100 mg·kg^-1 dose; higher doses could not be tested due to solubility constraints. Reflexive withdrawal from a noxious heat has not been associated per se with inflammation, although non-steroidal anti-inflammatory drugs can attenuate paw withdrawal in the hot plate assay. In the present study, KLS-13019 produced a significant increase in paw withdrawal latency starting at 1.0 mg·kg^-1. We were somewhat surprised at the level of antinociception observed with KLS-13019 in this assay; although the antinociceptive effect observed with the highest dose of KLS-13019 (100 mg·kg^-1) did not reach the full response produced by morphine (i.e., maximum latency of 25-s cut-off), a wider range of doses as well as potential mechanisms of action will be explored. Preliminary studies with KLS-13019 have revealed no motoric impairment effects as measured by rotarod performance, and we plan to reconfirm these results across a wider range of doses.

Currently, one of the most daunting challenges to treating pain safely and effectively is the fact that prescription opioids can be highly addictive. Mounting research focuses on whether cannabinoids can reduce or in some cases replace prescription opioid use for the treatment of chronic pain conditions. By extension, cannabinoids, including CBD, are specifically being investigated for their ability to reduce opioid craving in people with opioid use disorder (Hurd et al., 2019; Ren et al., 2009). To this end, we tested whether CBD or KLS-13019 could decrease motivated behaviour in rodent models of reinforcement. KLS-13019, but not CBD, significantly decreased progressive ratio responding for both a palatable food reinforcer and morphine. These results are preliminary, as our mouse morphine self-administration study was limited in scope to self-administration under a progressive ratio study with single doses of morphine and of CBD and KLS-13019. That said, our CBD results are consistent with the findings reported by Ren et al. (2009), who showed that CBD administration (5–20 mg·kg^-1) did not affect heroin intake in rats, extinction of heroin-seeking behaviour or prime-induced reinstatement behaviour, but only selectively decreased cue-induced heroin-seeking behaviour (5.0 mg·kg^-1). Our results do suggest that KLS-13019 can decrease reinforced behaviour. We are very intrigued by these results and again will explore these effects in a wider range of reinforcement assays and explore potential mechanisms by which KLS-13019 may attenuate the reinforcing effects of a palatable food reinforcer or morphine.

In conclusion, these studies demonstrate that like CBD, KLS-13019 can prevent the development of mechanical sensitivity following paclitaxel administration in mice, including following oral administration. Because KLS-13019, based on our previous data, binds to fewer biological targets, these findings can bring us closer to identifying molecular mechanisms shared by CBD and KLS-13019, which represent viable treatment targets for the prevention of the development of CIPN. While prevention of CIPN represents a significant unmet medical need, so does the treatment of existing CIPN for thousands of cancer survivors. The present results highlight the additional potential benefit of KLS-13019 in its ability to also reverse established paclitaxel-induced mechanical sensitivity, by extension underscoring the conclusion that KLS-13019 may work through unique mechanisms as compared with CBD in this model. Similarly, in the two additional antinociception models, KLS-13019 performs similarly to CBD at attenuated visceral pain in the stretching assay but outperforms CBD regarding attenuation of thermal nociception as measured by the hot plate. These antinociceptive effects of KLS-13019 are likely not mediated by interactions with opioid receptors as evidenced by the radioligand binding data. Lastly, KLS-13019 may possess the ability to attenuate reinforced behaviour, an effect not observed in the present study with CBD. Future studies will focus on identifying unique mechanisms of action for KLS-13019 in its ability to attenuate behaviours indicative of CIPN, visceral pain, thermal nociception and reinforced behaviour. Related to this, it will also be important to determine whether tolerance develops to the anti-nociceptive effects of KLS-13019. Lastly, KLS-13019 will be tested in drug discrimination and self-administration assays to determine it does not possess abuse liability on its own.

ACKNOWLEDGEMENT

Studies were supported by National Institutes of Health R41 DA044898-01 (SJW, DB Co-PIs).

AUTHOR CONTRIBUTIONS

Jeffery Foss, Daniel Farkas and Lana Huynh were involved in execution of behavioural testing and data analysis. William Kinney was responsible for overseeing compound synthesis. William Kinney, Douglas Brenneman and Sara Jane Ward were responsible for experimental design and manuscript preparation. Sara Jane Ward was responsible for overseeing data analysis and graphical reporting.

CONFLICT OF INTEREST

There are no competing interests to report.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Natural Products Research, Design & Analysis and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research.

Open Research

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.