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Low-Dose Cannabinoid Type 2 Receptor Agonist Attenuates Tolerance to Repeated Morphine Administration via Regulating µ-Opioid Receptor Expression in Walker 256 Tumor-Bearing Rats

Zhang, Mingyue MD; Wang, Kun MD; Ma, Min MD; Tian, Songyu MD; Wei, Na MD; Wang, Guonian MD

doi: 10.1213/ANE.0000000000001129
Anesthetic Pharmacology: Research Report

BACKGROUND: Morphine is widely used in patients with moderate and severe cancer pain, whereas the development of drug tolerance remains a major problem associated with opioid use. Previous studies have shown that cannabinoid type 2 (CB2) receptor agonists induce morphine analgesia, attenuate morphine tolerance in normal and neuropathic pain animals, induce transcription of the μ-opioid receptor (MOR) gene in Jurkat T cells, and increase morphine analgesia in cancer pain animals. However, no studies of the effects of CB2 receptor agonists on morphine tolerance in cancer pain have been performed. Therefore, we investigated the effect of repeated intrathecal (IT) injection of the low-dose CB2 receptor agonist AM1241 on the development of morphine tolerance in walker 256 tumor-bearing rats. We also tested the influence of the CB2 receptor agonist AM1241 on MOR protein and messenger ribonucleic acid (mRNA) expression in the rat spinal cord and dorsal root ganglia (DRG).

METHODS: Walker 256 cells were implanted into the plantar region of each rat’s right hindpaw. Tumor-bearing rats received IT injection of the CB2 receptor agonist AM1241 or antagonist AM630 with or without morphine subcutaneously twice daily for 8 days. Rats receiving drug vehicle only served as the control group. Mechanical paw withdrawal threshold and thermal paw withdrawal latency were assessed by a von Frey test and hot plate test 30 minutes after drug administration every day. MOR protein and mRNA expression in the spinal cord and DRG were detected after the last day (day 8) of drug administration via Western blot and real-time reverse transcription polymerase chain reaction. The data were analyzed via analysis of variance followed by Student t test with Bonferroni correction for multiple comparisons.

RESULTS: Repeated morphine treatments reduced the mechanical withdrawal threshold and thermal latency. Coadministration of a nonanalgetic dose of the CB2 receptor agonist AM1241 with morphine significantly inhibited the development of morphine tolerance and increased the MOR protein expression in the spinal cord and DRG and mRNA expression in the spinal cord in tumor-bearing rats.

CONCLUSIONS: Our findings indicate that IT injection of a nonanalgetic dose of a CB2 receptor agonist increased the analgesia effect and alleviated tolerance to morphine in tumor-bearing rats, potentially by regulating MOR expression in the spinal cord and DRG. This receptor may be a new target for prevention of the development of opioid tolerance in cancer pain.

Supplemental Digital Content is available in the text.Published ahead of print December 30, 2015

From the *Department of Anesthesiology, Cancer Hospital of Harbin Medical University, Harbin, China; Department of Gynecology, Cancer Hospital of Harbin Medical University, Harbin, China; and Department of Anesthesiology, Cancer Hospital of Harbin Medical University, Pain Research Institute of Heilongjiang Academy of Medical Sciences, Harbin, China.

Min Ma is currently affiliated with the Department of Anesthesiology, Inner Mongolia People’s Hospital, Hohhot, Inner Mongolia, China.

Accepted for publication November 5, 2015.

Published ahead of print December 30, 2015

Funding: This research was supported by funds from the Translational Medicine Special Foundation of China Russia Medical Research Center (no. 201519 and CR1418) and the Technological and Innovative Talent Foundation of Harbin (2012RFXXS041).

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website.

Reprints will not be available from the authors.

Address correspondence to Guonian Wang, MD, Department of Anesthesiology, Cancer Hospital of Harbin Medical University, No. 150 Haping Rd., Nangang District, Harbin 150081, China. Address e-mail to wangguonian609cn@aliyun.com.

Pain is the primary factor affecting the quality of life in patients with cancer. Despite the high risk of developing drug tolerance and dependence, morphine is a commonly used drug to manage moderate-to-severe pain.1,2 The analgesic effects of morphine are primarily mediated by the μ-opioid receptor (MOR).3 Previous studies have shown that prolonged exposure to opioid agonists reduces MOR gene expression and MOR-mediated G-protein activity,1,4,5 suggesting that downregulation of MOR expression could be associated with tolerance to chronic opioid treatment.

Synergistic interactions have been observed between cannabinoid (CB) and opioid analgesics.6,7 CBs are thought to predominantly exert their effects by binding to G-protein–coupled CB receptor type 1 and type 2 (CB2). The clinical efficacy of CBs acting at CB type 1 receptors is limited by central side effects and tolerance.8,9 CB2 receptors are considered to be peripheral and central receptors and have been detected in glia in distinct regions of the central nervous system such as the spinal cord and the dorsal root ganglia (DRG).10,11 Activation of the CB2 receptor induced analgesia in cancer pain12,13 and other experimental pain models.11,14,15 Some investigations have indicated that CB2 receptors are involved in morphine antinociception in normal and inflammatory conditions.16–18 In addition, several groups have reported that coadministration of low- or nonanalgetic-dose CB receptor agonists with morphine could reduce the morphine antinociceptive tolerance in normal rats and neuropathic pain animals.6,19,20 However, little is known about the effect of low-dose CB receptor agonists on morphine tolerance in cancer pain, especially the effect of CB2 receptor agonists on morphine tolerance.

In this study, we investigated whether a nonanalgetic dose of a CB2 receptor agonist could effectively reduce the morphine tolerance during cancer pain management. To achieve this, a nonanalgetic dose of the CB2 receptor agonist AM1241 and the selective CB2 receptor antagonist AM630 were injected intrathecally (IT) into the lumbar spinal cord 30 minutes before subcutaneous administration of morphine to assess the role of CB2 receptor agonists in the development of morphine tolerance in Walker 256 tumor-bearing rats. Furthermore, we quantified MOR protein and messenger ribonucleic acid (mRNA) expression in the rat spinal cord and DRG after chronic morphine treatment with or without a CB2 receptor agonist.

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METHODS

Animals

Adult (160–180 g) male Wistar rats (Yisi Experimental Animal Corporation, Changchun, China) were housed in a climate-controlled room in a 12-hour light/dark cycle and provided with food and water ad libitum. All experiments complied with the policies and recommendations of the International Association for the Study of Pain and were approved by the Animal Care and Use Committee of Harbin Medical University. After a week of habituation, each animal received Walker 256 tumor cell implantation on the plantar region of the right hindpaw. The Walker 256 cell culture and implantation method was performed as described previously.21

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Drug Administration

Drugs were given after 5 days of tumor cell inoculation. All drugs were administered twice daily (8:00 AM and 8:00 PM) for 8 days (day 1 to day 8). Repeated morphine was administered via subcutaneous injection of morphine sulfate (10 mg/kg/injection, 1 mL/kg volume, vehicle + morphine group, n = 10). Separate groups of animals received IT injection of a nonanalgetic dose of the selective CB2 receptor agonist AM1241 (0.07 μg; Cayman, Ann Arbor, MI) together with either morphine (AM1241 + morphine group, n = 10) or saline (AM 1241 + saline group, n = 10). Separate groups of animals received IT injections of the CB2 receptor antagonist (AM630; 10 μg; Cayman) along with AM1241 (0.07 μg) together with either morphine (AM1241 + AM630 + morphine group, n = 10) or saline (AM630 + saline group, n = 10). AM1241 and AM630 were diluted in dimethyl sulfoxide and saline at a ratio of 1:1 and were injected IT at a volume of 20 μL via lumbar puncture at the L5–L6 intervertebral space.11 Control animals received the same volume of vehicle and saline (vehicle + saline group, n = 10). The antagonist AM630 was injected 30 minutes before the agonist AM1241, and the agonist was injected 30 minutes before morphine.

The nonanalgetic dose of AM1241 we selected was based on our preliminary experiment and the nonanalgetic doses in bone cancer pain in mice (0.03 μg in hot plate test; 0.1 μg in von Frey test; IT) and neuropathic pain rats (0.01 μg in von Frey test; IT) in previous research.12,22 We tested 4 doses between 0 and 0.1 μg to determine whether they would be nonanalgetic doses in our animal model of cancer pain. The dose of 0.1 μg AM1241 induced analgesia in the von Frey and in hot plate tests compared with control, whereas the other 3 doses (0.03, 0.05, and 0.07 μg AM1241) had no statistical differences compared with the control (data not shown). Therefore, we chose 0.07 μg as the nonanalgetic dose of AM1241 in our study.

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Mechanical Hyperalgesia Test

Behavioral assays were performed before drug administration (at baseline) on the first test day and 30 minutes after drug administration each day in the morning during the drug treatment period. The withdrawal threshold to mechanical stimuli was assessed by applying von Frey filaments (Stoelting, Wood Dale, IL) and used a staircase method.23 Three measurements were performed on each animal randomly beginning with the left or right paw, and the average value was used.

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Thermal Nociception Test

Thermal withdrawal latencies were determined for each rat using a hot plate apparatus in a plastic cylinder (Technology & Market CORP, Chengdu, China). Rats were individually placed on the hot plate (52°C), and latency was defined as the time that elapsed before the rat licked a hindpaw or jumped. A threshold time of 30 seconds was set to prevent tissue damage. Three measurements were averaged to generate the final values.

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Western Blot Assays

Western blot experiments to detect the MOR protein expression were performed using lumbar segments of the spinal cord and DRG. After the behavioral tests on day 8, rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg) and were decapitated for tissue harvesting. Spinal lumbar L3/L4/L5 segments and DRG ipsilateral to the tumor cell injections were selected. Aliquots of total protein samples were analyzed using antibodies specific for MOR (1:1000; Abcam, Cambridge, United Kingdom); glyceraldehyde-3-phosphate dehydrogenase (1:500; ZSGB-BIO, Beijing, China) was used as a loading control. The immunoreaction was visualized using the ECL Plus chemiluminescence detection system and secondary antibodies (immunoglobulin G - horseradish peroxidase [IgG-HRP]; ZSGB-BIO).

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Real-Time Reverse Transcription Polymerase Chain Reaction

Real-time reverse transcription polymerase chain reaction (PCR) experiments to detect MOR mRNA expression were performed using lumbar L3/L4/L5 segments ipsilateral to the tumor cell injections from the spinal cord and DRG. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), and a 2-μg aliquot was used for complementary deoxyribonucleic acid (cDNA) synthesis using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). The cDNA was used as a template for real-time PCR amplification using Fast Start Universal SYBR Green Master Mix (Roche) and MOR primers (Invitrogen; forward primer, 5′-ACAGGCAGGGGTCCATAGAT-3′; reverse primer, 5′-GGATCGGCATGATGAAAGCG-3′) or glyceraldehyde-3-phosphate dehydrogenase primers (forward primer, 5′-AGATGGTGAAGGTCGGTGTG-3′; reverse primer, 5′-AACTTGCCGTGGGTAGAGTC-3′). PCR amplification was performed according to the manufacturer’s instructions (10 minutes at 95°C, 40 cycles of 10 seconds at 95°C, 30 seconds at 56°C, and 15 seconds at 95°C, 1 minute at 60°C, 30 seconds at 95°C, and 15 seconds at 60°C) using an ABI 7500 fast real-time PCR system (Applied Biosystems, Inc., Carlsbad, CA).

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Statistical Analysis

Power analysis was based on our results of a preliminary experiment of pain thresholds and molecular biological parameters. The sample size of each time point was calculated based on the observed SD in our preliminary experiment and yielded a sample size of n = 10 for pain thresholds and n = 5 for MOR protein and gene expression (α = 0.05; 1–β = 0.9) for each group. Detailed information of the preliminary experiment (the sample size, value of the SD, mean, days) is in the Supplemental Digital Content (Supplemental Tables 1–4, http://links.lww.com/AA/B343). Data were analyzed using SPSS 19.0 software (version 19.0; SPSS, Inc., Chicago, IL). For comparison of mechanical and thermal pain thresholds over time, 2-way analysis of variance for repeated measurements was used to examine the effects of treatment and time. If statistical main effects were observed (P < 0.01), the analysis was followed by Student t test with Bonferroni correction for multiple comparisons. MOR protein and mRNA expression levels were analyzed via a 1-way analysis of variance followed by Student t test with Bonferroni correction for multiple comparisons. We performed 7 comparisons at each period: vehicle + saline versus the other 5 groups, AM1241 + morphine versus vehicle + morphine, and AM630 + AM1241 + morphine versus vehicle + morphine. A P value less than the Bonferroni-corrected threshold of 0.0014 (0.01/7) was defined as statistically significant. Data are presented as the means ± SD.

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RESULTS

Development of Morphine Tolerance in Walker 256 Tumor-Bearing Rats

Figure 1

Figure 1

Figure 2

Figure 2

On days 1 and 2, morphine administration produced a significant analgesia compared with the vehicle + saline treatment based on mechanical hyperalgesia and thermal nociception (all P < 0.0001; Figs. 1 and 2). The level of morphine analgesia decreased on the consecutive days of chronic morphine treatment compared with day 1 (in von Frey test, day 3: P < 0.001; days 4–8, all P < 0.0001; in hot plate test, days 6–8: all P < 0.0001). Although on day 8 morphine consistently produced significant antimechanical hyperalgesia compared with vehicle + saline treatment (8.93 ± 1.43 vs 4.00 ± 1.70 g, P < 0.0001), this effect of morphine decreased significantly compared with its efficacy on day 1 (57.48 ± 5.00 vs 8.93 ± 1.43 g, P < 0.0001; Fig. 1). Thermal paw withdrawal latency on day 8 of chronic morphine administration did not differ statistically from that of vehicle + saline treatment (10.96 ± 3.18 vs 9.02 ± 2.22 seconds, P = 0.1328; Fig. 2), indicating that the rats receiving vehicle + morphine were tolerant to its analgesic effect of tumor-evoked pain.

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The Effect of AM1241 on the Development of Morphine-Mediated Mechanical Hyperalgesia

The combination of AM1241 and morphine (AM1241 + morphine group) strengthened analgesia of morphine for mechanical stimulus on days 7 and 8 (P < 0.0001) compared with morphine administration alone (Fig. 1). Although mechanical withdrawal thresholds of AM1241-pretreated rats (AM1241 + morphine group) were reduced substantially from day 4 to day 8 compared with day 1 (days 4–8: all P < 0.0001), their analgesic response remained significantly higher than the animals injected with morphine alone on days 7 and 8 (AM1241 + morphine group versus vehicle + morphine group, P < 0.0001). The selective CB2 receptor antagonist AM630 reversed the effects of AM1241 on morphine analgesia and tolerance and displayed no difference between the AM630 + AM1241 + morphine group and vehicle + morphine group (all P > 0.1249). The analgesic response of repeated administration of AM1241 alone (AM1241 + saline group, 0.07 μg, IT) did not differ from the vehicle + saline group. Detailed information of multiple comparisons for mechanical withdrawal threshold (mean difference, 99% confidence interval, and P value) can be found in the Supplemental Digital Content (Supplemental Tables 5 and 6, http://links.lww.com/AA/B343).

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The Effect of AM1241 on Development of Morphine-Mediated Thermal Nociception

The combination of AM1241 and morphine (AM1241 + morphine group) strengthened the analgesia of morphine for thermal nociception on days 6, 7, and 8 (all P < 0.0001) compared with administration morphine alone (Fig. 2). Although the paw withdrawal latency of AM1241-pretreated rats (AM1241 + morphine group) decreased on day 8 compared with day 1 (P < 0.0001), the analgesic response to morphine on day 8 remained significantly greater (21.09 ± 2.61 vs 10.96 ± 3.18 seconds, P < 0.0001) in the AM1241-pretreated rats compared with the morphine alone–injected group, which displayed tolerance to morphine analgesia. The latency values were not statistically different from vehicle + saline-treated rats (10.96 ± 3.18 vs 9.02 ± 2.22 seconds; P = 0.1328). No difference was observed in the analgesic response of the AM630 + AM1241 + morphine group compared with the morphine-injected group in the hot plate test. The analgesic response of repeated administration of AM1241 alone (AM1241 + saline group, 0.07 μg, IT) did not differ from the vehicle + saline group. Detailed information of multiple comparisons for thermal withdrawal latency (mean difference, 99% confidence interval, and P value) can be found in the Supplemental Digital Content (Supplemental Tables 7 and 8, http://links.lww.com/AA/B343).

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The Effect of AM1241 on the Morphine-Induced MOR Protein Expression in the Spinal Cord and the Lumbar DRG

Figure 3

Figure 3

Western blot analysis revealed a band at approximately 55 kDa corresponding to the MOR in samples obtained from the spinal cord (Fig. 3, A and C) and DRG (Fig. 3, B and D). The vehicle + saline group served as the control group in Figure 3. AM1241-pretreated rats displayed a significant increase in MOR expression in the spinal cord (P < 0.0001) and DRG (P < 0.0001) compared with morphine administration alone. The effect of AM1241 on morphine-induced MOR protein expression was abolished by the selective CB2 receptor antagonist AM630, and there was no difference between the AM630 + AM1241 + morphine group and vehicle + morphine group (P = 0.6541 in spinal cord; P = 0.2427 in DRG). Detailed information of multiple comparisons for MOR protein expression (mean difference, 99% confidence interval, and P value) can be found in the Supplemental Digital Content (Supplemental Table 9, http://links.lww.com/AA/B343).

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The Effect of AM1241 on the Morphine-Induced MOR mRNA Expression in the Spinal Cord and the Lumbar DRG

Figure 4

Figure 4

The vehicle + saline group served as the control group in Figure 4. There was no significant difference in MOR mRNA expression among the 6 groups in the L3/L4/L5 DRG (P = 0.0447). Pretreatment with AM1241 significantly increased MOR mRNA expression in the spinal cord compared with morphine administration alone (P < 0.0001). Pretreatment with AM630 significantly reversed the effect of AM1241 on morphine-induced MOR mRNA expression, and there was no difference between AM630 + AM1241 + morphine group and vehicle + morphine group on MOR mRNA expression in the spinal cord (P = 0.8066). The detailed information of multiple comparisons for MOR mRNA expression (mean difference, 99% confidence interval, and P value) can be found in the Supplemental Digital Content (Supplemental Table 10, http://links.lww.com/AA/B343).

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DISCUSSION

In the present study, we found that coadministration of a nonanalgetic dose of the CB2 receptor agonist AM1241 with morphine reduced tolerance to the analgesic effects of morphine, increased MOR protein expression in the spinal cord and DRG, and mRNA expression in the spinal cord of tumor-bearing rats. Our findings strongly suggest that a CB2 receptor agonist may play a positive role in attenuating morphine tolerance in cancer pain treatment.

Several studies have shown that coadministration of a low- or nonanalgetic-dose CB circumvented antinociceptive tolerance to morphine in normal rats and animals models of neuropathic pain.6,19,20 However, the effect of a low-dose CB2 receptor agonist on morphine tolerance in cancer pain has not been reported. Our findings indicated that IT administration of a nonanalgetic dose of the CB2 receptor agonist AM1241 increased the analgesic effects of morphine while alleviating morphine tolerance during the treatment of cancer pain. There are several reasons for these results that should be considered. First, the selective CB2 receptor agonist attenuates morphine tolerance via upregulating MOR expression in cancer pain. However, it is possible that the CB2 receptor agonist alleviates morphine tolerance by an interaction between opioid and CB receptors and by reduction of glial and mitogen-activated protein kinase (MAPK) activation.7,24

MOR expression was different in the dorsal horn and DRG in various animal models.25–27 MOR expression was decreased in the ipsilateral DRG based on a neuropathic and bone cancer pain model,25,26 thereby reducing the effects of MOR agonists.28,29 Here, we observed that coadministration of AM1241 with morphine led to an increase in MOR protein expression both in the spinal cord and DRG after chronic morphine exposure in this Walker 256 tumor-bearing animal model. Furthermore, we detected the level of MOR mRNA in the spinal cord and DRG to judge whether upregulation of MOR expression happened in transcription. As we expected, the level of MOR mRNA was increased in the spinal cord of AM1241-pretreated rats, but not in the DRG. In this cancer pain model, the reason for the different effects of AM1241 on mRNA expression between the spinal cord and DRG was not clear. The present results suggest that CB2 receptor agonists could attenuate morphine tolerance via regulating MOR expression in cancer pain.

Although the role of CBs in opioid tolerance has been studied, the mechanism underlying these actions has not been defined. Opioid and CB receptors both belong to the family of Gi/Go-protein–coupled receptors and are often coexpressed in the cells of the nervous system.30 The interaction between opioid and CB receptors may be facilitated by similar intracellular signal pathways.2,24 Previous studies have shown that expression and activation of MOR in the brainstem are attenuated by the CB2 receptor antagonist SR144528 via CB2 receptors.31 The CB2-specific antagonist AM630 inhibited MOR expression, whereas the CB2-specific agonist JWH 015 markedly induced MOR expression in Jurkat T cells.32 These regulatory events were mediated by a signal transducer and activator of transcription (STAT)5-interleukin (IL)-4-STAT6 signaling pathway.32 Expression of IL-4 has also been detected in astrocytes from multiple sclerosis lesions33 and in lipopolysaccharide-activated microglia.34 In addition, IL-4 induces MOR transcription in primary neurons.35 These findings indicate that CBs induce MOR expression via an IL-4-dependent pathway in neurons of the central nervous system.

Opioids and CBs may interact at multiple levels and have been associated with analgesic, psychotrophic, and immunomodulatory effects. Previous studies demonstrated that the activation of glial cells by chronic morphine administration contributes to morphine tolerance.2,18,36 CB2 receptors have been detected in glial cells in distinct regions of the nervous system, such as the spinal cord and DRG.10,11 Glial CB2 receptors are dramatically upregulated in response to damaging stimuli11,37 and opioid agonist treatment.7,16 The CB2 receptor agonist attenuated inflammatory and neuropathic activation of glia in the central nervous system11,38 and coadministration of the selective CB2 receptor agonist AM1241 with morphine reduced morphine-mediated activation of spinal glia.7 MAPK activation2 and glial proinflammatory mediator release39,40 have also been linked to morphine tolerance. Administration of the CB2 receptor agonist reduces MAPK phosphorylation in neuropathic pain24 and morphine-induced inflammatory responses in activated microglial cells.18 Based on these findings, we speculate that there are possible mechanisms by which CB2 receptor agonists attenuate morphine tolerance in cancer pain.

There are some limitations to our study. First, we only used a single dose of a CB2 receptor agonist with morphine to determine the role of a CB2 receptor agonist in morphine tolerance. It would have been much more useful to know the effects over a range of agonist doses. Second, this study used measurement of MOR protein and mRNA expression at a single time point, that is, on day 8, did not determine MOR expression on other tested days. Third, we did not investigate the pathway between the CB2 receptor and MOR. Thus, the cellular and neurophysiologic pathways involved in the development of CB-related morphine tolerance remain uncertain in cancer pain. Further studies are required to address these limitations.

In summary, the present results suggest that CB receptor agonists are involved in opioid-mediated analgesia and attenuate opioid tolerance during management of cancer pain. Upregulation of MOR expression by CB2 receptor agonists may contribute to the synergistic effects of CBs and opioids. The use of CBs for cancer treatment is currently limited to chemotherapy- or radiotherapy-associated nausea.41 Our current data provide a novel pharmacologic approach using CB2 receptor agonists to strengthen morphine analgesia, thereby reducing the doses and side effects of morphine used for severe pain therapies.

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DISCLOSURES

Name: Mingyue Zhang, MD.

Contribution: This author helped design the study, analyze the data, and prepare the manuscript.

Attestation: Mingyue Zhang approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author.

Name: Kun Wang, MD.

Contribution: This author helped analyze the data.

Attestation: Kun Wang approved the final manuscript.

Name: Min Ma, MD.

Contribution: This author conducted the study and helped in data collection.

Attestation: Min Ma approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

Name: Songyu Tian, MD.

Contribution: This author helped with data collection

Attestation: Songyu Tian approved the final manuscript.

Name: Na Wei, MD.

Contribution: This author helped with data collection.

Attestation: Na Wei approved the final manuscript.

Name: Guonian Wang, MD.

Contribution: This author helped design the study and prepare the manuscript.

Attestation: Guonian Wang approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.

This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.

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ACKNOWLEDGMENTS

We thank professors Yuyan Ma and Hongbo Jin (Cancer Institute of Heilongjiang Province and Department of Physiology, Harbin Medical University) for their assistance with this project. We thank the analyst Shaofei Su (Department of Statistics of Harbin Medical University) for help with statistical analyses.

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