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. 2020 Feb 19;185(Suppl 1):130–135. doi: 10.1093/milmed/usz320

Chemokine Receptor Antagonists in Combination with Morphine as a Novel Strategy for Opioid Dose Reduction in Pain Management

Toby K Eisenstein 1, Xiaohong Chen 1, Saadet Inan 1, Joseph J Meissler 1, Christopher S Tallarida 1, Ellen B Geller 1, Scott M Rawls 1, Alan Cowan 1, Martin W Adler 1
PMCID: PMC7353838  PMID: 32074321

Abstract

Introduction

Although opioids are widely prescribed for pain, in many circumstances, they have only modest efficacy. Preclinical studies have shown that chemokines, immune mediators released during tissue injury and inflammation, can desensitize opioid receptors and block opioid analgesia by a process termed “heterologous desensitization.” The present studies tested the hypothesis that in evoked pain, certain chemokine receptor antagonists (CRAs), given with a submaximal dose of morphine, would result in enhanced morphine potency.

Methods

Three rodent pain assays were used: incisional pain in rats, the cold-water tail flick test in rats, and the formalin test in mice. The FDA-approved, commercially available CRAs, maraviroc and AMD3100, were used. They block the chemokine receptors and ligands, CCR5/CCL5 (RANTES) and CXCR4/CXCL4 (SDF-1α), respectively.

Results

In the incisional pain assay, it was found that the combination of a single CRA, or of both CRAs, with morphine significantly shifted the morphine dose-response curve to the left, as much as 3.3-fold. In the cold-water tail flick and formalin tests, significant increases of the antinociceptive effects of morphine were also observed when combined with CRAs.

Conclusions

These results support the potential of a new “opioid-sparing” approach for pain treatment, which combines CRAs with reduced doses of morphine.

INTRODUCTION

Pain management continues to be a major medical problem for treatment of personnel in the armed forces, as well as in the civilian population. Overuse of opioids is currently a major problem of epidemic proportion, with 47,600 deaths in 2017.1 According to a 2017 Substance Abuse and Mental Health Services Administration report, 63% of people who misused opioids gave relief of pain as the major reason for taking the drugs.2 It is well recognized that opioids are less effective in chronic pain, which usually involves an underlying inflammatory component.3 Chemokines are a family of 41 small protein molecules released by a variety of immune cells that increase inflammatory responses by attracting subsets of leukocytes that express the cognate chemokine receptor, which leads to a process of directed cell migration to the site of inflammation. Receptors for opioids and chemokines are members of the G-protein-coupled seven-transmembrane family. Several studies have established an interaction between chemokines and opioids via heterologous cross-desensitization of their receptors. Grimm et al4 showed that the directed migration of human peripheral blood mononuclear cells, exposed to a chemokine in vitro, was blocked by prior exposure to the opioid peptide, met-enkephalin. Further, chemokines were shown to desensitize mu opioid receptors on dorsal root ganglia neurons.5 Our laboratories have also provided evidence that chemokines can desensitize opioid receptors in the neural system. Rats, cannulated in the pain-sensing periaqueductal gray (PAG) region of the brain, responded to infusion of morphine by keeping their tails immersed in ice-cold water for a longer time (cold-water tail flick [CWTF] test). Infusion of a chemokine up to half an hour prior to giving morphine-blocked morphine-induced analgesia.6 Further, single neurons of the PAG express both chemokine receptors and opioid receptors.7 Our group has also shown that transfected cells can form heterodimers between opioid and chemokine receptors, and the fact that both receptors can be detected on single neurons makes the potential for interaction between these two classes of receptors feasible in the nervous system.8 Electrophysiological tracings of PAG neurons have also demonstrated an interaction between chemokines and morphine.7 These studies provide a mechanism to explain why in many pain conditions opioids are less effective. If chemokines are released as part of the inflammatory process, they could desensitize opioid receptors, making them less responsive to morphine and other opioids. Based on these observations, the hypothesis was formulated that chemokine receptor antagonists (CRAs), by blocking the signaling ability of chemokines at their receptors, would augment opioid potency and provide greater analgesia for a given dose of an opioid. In these studies, a submaximal dose of morphine was tested in combination with a CRA, or two CRAs, in several pain assays. This strategy was found to be effective in enhancing the potency of morphine in such assays.

METHODS

Animals

Male Sprague-Dawley rats (Taconic Biosciences, Germantown, New York), weighing 175–200 g, were housed in groups of 2, and Swiss-Webster female mice (Taconic Biosciences), weighing 15–20 g, were housed in groups of 5. All animals were acclimated for at least 1 week in an animal room maintained at 22 ± 1°C and approximately 50 ± 5% relative humidity. Lighting was on a 12/12 h light/dark cycle. All animals were allowed free access to food and water. Animal care and experimental procedures were approved and performed in accordance with the Institutional Animal Care and Use Committee of Temple University.

Drugs

Morphine sulfate was obtained through the NIDA Drug Supply Program and was dissolved in 0.9% saline. The chemokine antagonists, AMD3100 (dissolved in sterile, pyrogen-free water) and maraviroc (dissolved in 5% DMSO for mice or 10% DMSO for rats) were obtained from Sigma-Aldrich, Inc. (St. Louis, Missouri). AMD3100 binds to the chemokine receptor, CXCR4, and blocks the chemokine, CXCL12/SDF-1α.9 Maraviroc selectively binds to CCR5 and blocks the chemokine, CCL5/RANTES.10,11

Pain Assessment Assays

Three pain models were used in the present study to test the analgesic potency of morphine alone, or in combination with CRAs.

Incisional Pain Assay in Rats

In this assay, a 1.0-cm longitudinal incision was made with a scalpel through the skin and fascia of the plantar surface of the rat left hind paw, and the post incisional sensitivity to mechanical allodynia was measured in control and treated animals using von Frey filaments. The details of this assay are as follows: 2 days before surgery and behavioral testing, rats were acclimated in individual transparent cubicles with a wire mesh floor for an hour each day. On the day of surgery, rats were acclimated for 30 min and then their individual baseline values for paw withdrawal threshold were measured using a series of von Frey filaments (North Coast Medical, Inc., Gilroy, California), with gradually increasing equal logarithmic bending forces (spanning forces of 2–60 g). The filaments were applied to the plantar side of each hind paw in an ascending manner. Each filament was tested five consecutive times a few seconds apart. A positive response was defined as quick withdrawal or paw flinching after the application of a filament. Surgery was performed as previously described by Brennan et al.12 under isoflurane anesthesia (4% isoflurane for induction and 2.5% isoflurane for maintenance of anesthesia) in aseptic conditions. The time when surgeries were completed was designated as time 0. The animals awoke on the average 5–8 min after the end of the surgery, and fully regained consciousness by the first testing time, which was 15 min post surgery. Morphine, CRAs, or their vehicles were injected subcutaneously (s.c.) at t = 25 min post surgery into separate sites on the right or left flank, as well as behind the neck area if needed for a third injection. Paw withdrawal thresholds were recorded at various time points (15–360 min) post surgery by a person blinded to the treatments. Percent reversal of mechanical allodynia was calculated at the 60 min time point using the formula:

Inline graphic The test threshold represents paw pressure response to the filament at testing time point; the predose threshold represents the response at 15 min after surgery before treatments; and the baseline threshold represents the baseline value before surgery. The percentage reversal of mechanical allodynia was calculated individually for each rat and then the mean, SD, and SEM values were calculated for individual groups.

Cold-Water Tail Flick Test in Rats

The latency to flick the tail in cold water was used as the antinociceptive index, according to a standard protocol.13 A 1:1 mix of ethylene glycol:water was maintained at −3°C in a circulating water bath. Rats were held over the bath with their tails submerged approximately halfway into the solution. All animals were tested at 60, 15, and 0 min before drug injection. For each animal, the first reading was discarded and the mean of the second and third readings was taken as the baseline value. Latencies to tail flick after injection of drugs were expressed as percentage change from baseline. Measurements were taken at 30, 45, and 60 min after injection of vehicle or drugs. A cutoff limit of 60 sec was set to avoid damage to the tail. The percent of maximal possible antinociception (%MPA) for each animal at each time was calculated using the following formula: %MPA = [(test latency—baseline latency)/(60—baseline latency)] × 100. To test the effect of morphine, with or without administration of CRAs, the following protocol was used. At t = 0, rats were injected s.c. with either morphine or vehicle on one dorsal flank. On the opposite flank, they received a s.c. injection of vehicle or a CRA. If both AMD3100 and maraviroc were used in the same experiment, the two CRAs were injected on the opposite flanks. Control animals received only vehicle injections. Tail withdrawal determinations were recorded by a person blinded to the treatments.

Formalin Pain Assay in Mice

The standard formalin paw injection assay was used.14 A solution of 5% formalin prepared in 0.9% saline was injected into the dorsal surface of the right hind paw of Swiss-Webster, female mice (25–35 g) in a volume of 20 μL. Morphine, CRAs, or vehicle was injected into separate s.c. sites on the right and left flanks. Mice were then placed in a transparent box for observing the licking response. At t = +20 min post formalin injection, the duration time of formalin-injected paw licking was scored for the next 15 min (licking that occurred between 20 and 35 min post formalin injection). Licking was scored by a person blinded to the treatment.

Quantitation of mRNA for Immune Mediators

For the experiments using incisional pain, the effect of morphine alone, CRAs alone, or the combination of morphine plus the CRAs, on levels of mRNA of selected immune mediators, was determined using the TaqMan Gene Expression Assays (Applied Biosystems, ThermoFisher, Foster City, California). Paw tissue samples were collected from baseline animals (no surgery), animals given the incision surgery with no treatment, and rats given surgery plus morphine alone, the CRAs alone, or morphine plus the CRAs. Paw tissues were collected 1 hr post incision by excision and flash freezing to −80°C. Tissues were processed by the Qiagen RNeasy Microarray Tissue Mini Kit (Qiagen, Germantown, Maryland) following the manufacturer’s protocols. Individual mRNA preparations were quantitated by a Nanodrop 2000 spectrophotometer (ThermoScientific, Waltham, Massachusetts) to determine mRNA concentrations in ng/μl. mRNAs from paw tissue of three individual rats from each treatment group were used to generate cDNAs using the SuperScript III First-Strand Synthesis System for RT-PCR (inVitrogen, ThermoFisher, Carlsbad, California), which were then assayed using the TaqMan Gene Expression Assays for rat CXCL1, CXCL2/MIP-2, CCL2/MCP-1, CCL5/RANTES, CXCL12/SDF-1α, and β2-microglobulin. Each gene expression assay was tested on all three samples from each treatment group. The reactions were performed using TaqMan Fast Advanced Master Mix. The RT-PCR array was run on an Applied Biosystems StepOne Plus RT-PCR thermocycler, using the cycling conditions for TaqMan reagents. Results obtained are expressed as the mean log2-fold difference in mRNA expression in the treated groups compared with the level of mRNA expression in the baseline untreated control. Data were processed by the DataAssist v3.01 software (Applied Biosystems).

Statistical Analysis

The size of groups of animals used in the pain tests varied from 7 to 10. Power analysis determined 6 animals/group to be sufficient. Where the power is 80%, alpha is 0.05, beta is 0.2, incidence for group 1 is 10% and for group 2 is 90%. Data for the results of pain tests are expressed as the mean +/− the standard error of the mean. Differences between groups were assessed using either one- or two-way analysis of variance (ANOVA) depending on the number of variables. The Sidak’s multiple comparison test was applied if the ANOVA was p < 0.05 in order to test whether morphine alone vs morphine plus a CRA was significant. The data were analyzed using Prism Graphpad software version 7. In all cases, p < 0.05 was taken as the significant level of difference.

RESULTS

Incisional Pain Test in the Rat

These experiments sought to test whether the combination of one or two CRAs with a submaximal dose of morphine could result in maximal analgesia. Neither CRA produced significant analgesia by itself in the incisional pain test.15 The analgesic activity of the combination of various doses of morphine plus either AMD3100 (6.7 mg/kg) or maraviroc (5.0 mg/kg) and of morphine in combination with both CRAs was tested. The composite results of von Frey measurements taken at 60 min are shown in Figure 1. The combination of morphine with a single CRA significantly shifted the morphine dose-response curve to the left (ED50 for morphine alone = 2.34 mg/kg; ED50; for morphine + AMD3100 = 1.32 mg/kg, a 1.8-fold shift; ED50 for morphine + maraviroc = 1.01, a 2.3-fold shift). The ED50 of the combination of morphine plus both CRAs was 0.71 mg/kg, a 3.3-fold shift to the left compared to morphine alone.

Figure 1.

Figure 1

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Combination of morphine with 2 CRAs in the incisional pain test shifts the morphine dose-response curve to the left. Rats were injected with morphine alone or with the CRAs, into separate s.c. dorsal sites 25 min after completion of the surgery. Percent reversal of mechanical allodynia was calculated using data at 60 min post incision. The combination of morphine with either AMD3100 or maraviroc, individually, as well as the combination of morphine with both CRAs, significantly shifted the morphine dose-response curve to the left. The paired Student t-test showed *p < 0.05, (n = 6–10). (From: S Inan, TK Eisenstein, MN Watson, M Doura, JJ Meissler, CS Tallarida, X Chen, EB Geller, SM Rawls, A Cowan, and MW Adler (2018). Coadministration of Chemokine Receptor Antagonists with Morphine Potentiates Morphine’s Analgesic Effect on Incisional Pain in Rats. J Pharmacol Exp Ther. 367 (3): 433–44. (Used with permission).

mRNA Levels in Paw Tissue in the Incisional Pain Test

As shown in Figure 2, mRNAs for the 5 chemokines analyzed were detected in paw tissue 1 hr after incision. mRNA for CCL5 and CXCL12, the chemokines that are blocked by maraviroc and AMD3100, respectively, did not show an elevation as a result of the incision. The other 3 chemokines, which are involved in acute inflammatory responses, CXCL1, CXCL2 and CCL2, were higher than baseline, but their increase was only a little over 2-fold. Although none of the results were statistically significant for these 3 chemokines, the CRAs alone, or the combination of the CRAs with the suboptimal dose of morphine, shifted mRNA levels to below baseline.

Figure 2.

Figure 2

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Fold difference of gene expression in rat paw tissue, from animals receiving incision and morphine plus CRAs. Data are expressed as log2-fold difference in expression compared with untreated control rat samples. Data are presented as the mean level of mRNA expression in paw tissue from 3 individual animals. Dotted lines indicate limits of 2-fold up- or down-regulation from baseline.

Cold-Water Tail Flick Test in the Rat

A similar series of experiments was carried out for the CWTF test as was done for the incisional pain test described above. Individual dose-response and time curves were obtained for AMD3100 and maraviroc, each tested individually, for analgesic activity. Neither CRA had any intrinsic antinoceptive activity in this test (data not shown). Morphine alone gave a typical dose-response curve with significant analgesia (83% of the MPA) observed at all time points for the 10 mg/kg dose, and lesser analgesia observed with lower doses. Pain thresholds were measured at 30, 45, and 60 min, but only the results that showed maximal statistical significance are presented in Figure 3, which was at 30 min for maraviroc and 45 min for AMD3100. When the 5 mg/kg dose of morphine was combined with maraviroc (5 mg/kg), significant enhancement of analgesia was observed compared to morphine alone at the 30-minute time point (Fig. 3A). The combination of morphine (3 mg/kg) and AMD3100 (7.5 mg/kg) produced a significant enhancement of the MPA compared to morphine alone at the 45 min time point (Fig. 3B).

Figure 3.

Figure 3

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Effects of the combination of a submaximal dose of morphine plus a chemokine in the cold-water tail flick test. Panel A: Percent maximal possible analgesia of different doses of morphine in combination with AMD3100. Measurement at 30 min post drug or vehicle injection. Panel B: Percent maximal possible analgesia of different doses of morphine in combination with maraviroc. Measurement at 45 min post drug or vehicle injection. Two-way ANOVA followed by Sidak’s multiple comparison test showed significant differences between morphine alone vs. morphine plus a CRA. *p < 0.05.

Formalin Test in the Mouse

As in the other two pain tests described above, neither AMD3100 nor maraviroc alone displayed analgesic activity (data not shown). When a submaximal dose of morphine (1 mg/kg) was combined with either AMD3100 (5 mg/kg) or maraviroc (5 mg/kg), significantly enhanced analgesia was observed compared to morphine alone, as evidenced by decreased paw-licking time (Fig. 4A and B).

Figure 4.

Figure 4

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Effect of combinations of morphine with AMD3100 (panel A) or maraviroc (panel B) on analgesia in the mouse formalin test. One-way ANOVA followed by Sidak’s multiple comparison test showed significant differences between morphine alone and either morphine plus AMD3100 (panel A) or morphine plus maraviroc (panel B). *p < 0.05.

DISCUSSION

The relationship of various chemokines to several pain states has been reported.16–18 Our laboratories reported that CCL5 induced hyperalgesia in the CWTF test, and an antibody to this chemokine blocked the effect.19 Various pain stimuli have been shown to be reduced in CCR5 knockout mice.20,21 Pain associated with administration of antiretroviral drugs that induce toxic neuropathy, alone or in combination with HIV gp120, has been shown to be mediated by up-regulation of CXCL12, and blocked by treatment with a CRA, AMD3100.22,23 A synthetic dual CRA, RAP-103, that targets CCR5 and CCR2, attenuated neuropathic pain in rats.21 A bivalent ligand that is a mu opioid agonist and a CCR5 antagonist produced nociception.24 The concept of using a CRA in conjunction with reduced doses of morphine for pain control emanated from experiments that had been conducted in our laboratories, and confirmed by others, showing that selective chemokines blocked morphine analgesia via a process termed “heterologous desensitization.”5,6 These observations led to the formulation of the hypothesis that inhibition of chemokines could augment the potency of morphine. In this article, data are presented using three different pain assays in rodents to support this hypothesis. It was shown in all three tests that submaximal doses of morphine, when combined with a single CRA, produced analgesia that was significantly greater than that observed with morphine alone. In the incisional test, the combination of morphine plus 2 CRAs was tested and found to be superior to that of morphine plus a single CRA. Results were most robust in the incisional pain assay where the compounds were given therapeutically after surgery. Morphine plus AMD3100 and maraviroc shifted the morphine dose-response curve an impressive 3.3-fold to the left. In the CWTF test and the formalin pain test, significant increases in antinociception were also found using selected submaximal doses of morphine plus a CRA. The mechanism by which the CRAs are exerting this additive effect with morphine is hypothesized to be by blocking the heterologous desensitization of morphine receptors by the chemokines. Based on this theory, it was expected that the combination treatment would lower the levels of CCL5 and CXCL12. The results in Figure 2 show that mRNA for these chemokines, that are the cognate ligands of the CRA inhibitors that were used, were not elevated. These individual PCR results corroborate data obtained using an mRNA array.15 The mechanism by which the morphine plus CRA therapy lowers mRNA for chemokines that are not part of the combination administered is not currently understood.

Of the three pain assays presented in this article, two were carried out in male rats, the incisional pain test and cold-water tail flick test. The studies for the formalin test were carried out in female mice. This article does not attempt to make a conclusion about differences between sexes in response to the pain assays or treatment. The only assay where both male and female animals were used was the formalin assay. Because the results in male animals were more variable than what was observed in female mice, only the results for the females are presented in this manuscript.

As part of these studies, the paradigm of combining a suboptimal dose of morphine with one or more CRAs was also tried in a carrageenan rat pain model (resulting in both pain and edema) where the substance is injected into the paw, and paw withdrawal thresholds tested using a heat beam, and in a rat paclitaxel model of chemotherapy-induced neuropathic pain assessed by mechanical allodynia using von Frey filaments. The combination therapy of morphine plus CRAs was not effective in either of these pain tests. The choice of preclinical pain tests to best simulate human pain conditions is complex, and different mechanisms may be involved in different types of pain.3

In the present study, the combination therapy of a submaximal dose of morphine in combination with one or more CRAs was efficacious in three different pain assays. These results represent a new approach to pain treatment that has potential for use in a variety of acute and chronic pain conditions resulting from surgery or injury. This opioid-sparing strategy might have the additional advantage of reducing opioid-induced side effects such as respiratory depression and inhibition of gastrointestinal transit, as morphine could be used in markedly lower doses. Experiments are in progress to explore if these morphine-CRA combinations do in fact result in diminished undesirable effects of opioids.

ACKNOWLEDGMENTS

We thank Mia N. Watson, B.Sc., Menahem Doura, Ph.D., and Emily Dziedowiec, B.S., for excellent technical assistance.

Guarantor: Martin W. Adler

Military Health System Research Symposium, Kissimmee, FL, August 20-13, 2018. Oral presentation. Abstract # MHSRS-18-0804. International Narcotics Research Society meeting, San Diego, CA, June 14-17, 2018, Poster#32. College on Problems of Drug Dependence meeting, San Diego, CA, June 9-14, 2018, Poster#26. International Narcotics Research Society meeting, Chicago, IL, July 9-14, 2017, Poster#42.

FUNDING

Department of Defense grant # W81XWH-15-1-0252 and NIH/NIDA P30 DA013429.

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