Activation of Peripheral μ-opioid Receptors by Dermorphin [d-Arg2, Lys4] (1–4) Amide Leads to Modality-preferred Inhibition of Neuropathic Pain

CHRONIC neuropathic pain is prevalent among 6 to 8% of the population. In patients attending pain clinics, the incidence is as high as 25 to 51.9%.¹  Moreover, chronic pain causes considerable social and economic burden and leads to high healthcare costs and lost productivity.^2,3  Patients with neuropathic pain resulting from nerve injuries present with varying degrees of mechanical and heat hyperalgesia. These manifestations are also observed in animal models of neuropathic pain.^4,5  Mechanical and heat hypersensitivities involve different peripheral and central mechanisms and hence may require different treatment strategies.^4,6,7  In the past several years, a number of drugs have been developed for the treatment of neuropathic pain, but no single agent is uniformly effective, and opioids remain some of the most commonly used drugs. However, their therapeutic utility is limited considerably by their severe central and peripheral side effects, including sedation, dizziness, respiratory depression, constipation, nausea, vomiting, tolerance, and physical dependence.⁸  These adverse effects may lead to opioid discontinuation and contribute to underdosing and inadequate analgesia in patients with neuropathic pain.⁹  The potential adverse effects are a primary reason that opioids have been downgraded from a second-line class to a third-line class of drugs in the recent recommendations for the pharmacologic treatment of neuropathic pain.¹⁰  Hence, alternative therapies that lack central and peripheral adverse effects are important for the treatment of neuropathic pain.

Activating opioid receptors in the peripheral nervous system may offer an opportunity for treating certain chronic pain conditions while avoiding their central side effects.^11,12  However, whether peripheral opioids affect mechanical and heat hypersensitivity differently is unclear. In addition, little is known about their cellular mechanisms, such as which subpopulations of primary sensory neurons are targeted by peripherally acting opioids to inhibit different neuropathic pain modalities. Dermorphin is a natural heptapeptide μ-opioid receptor (MOR) agonist found in amphibian skin.^13,14  Degradation of dermorphin by peptidases produces the N-terminal tetrapeptide H-Tyr-d-Ala-Phe-Gly-OH.^14,15  Additional amino acid substitutions to this tetrapeptide led to the development of dermorphin [d-Arg2, Lys4] (1–4) amide (DALDA), a highly selective MOR agonist.^16–18  Intriguingly, DALDA is highly hydrophilic because it carries three net positive charges at physiologic pH. Moreover, it is metabolically more stable than its other analogs and exhibits a restrictive penetration into the central nervous system (CNS) after systemic drug administration.¹⁷  These properties make DALDA a promising drug candidate for the treatment of chronic neuropathic pain with reduced risk of central side effects.

Critical to the use of peripherally acting opioids is an understanding of the analgesic properties and mechanisms that underlie the therapeutic effects of DALDA. Therefore, the purpose of this research was to improve our understanding of the cellular mechanisms involved in the peripheral opioid analgesia and the development of peripheral opioids with minimal central side effects. In this study, we characterized the efficacy of systemic DALDA to attenuate mechanical and heat hypersensitivity in nerve-injured rats, investigated its site and mechanism of action, and assessed its safety profile.

Male and female Sprague-Dawley rats (200 to 350 g; Harlan Bioproducts for Science, USA) were housed under optimal laboratory conditions with a 12-h light/dark cycle and free access to food and water. Animals were acclimatized to laboratory conditions before the tests. All behavioral experiments were carried out between 9:00 am and 5:00 pm by an investigator blinded to the drug assignment. The experimental protocols were approved by the Animal Care and Use Committee of Johns Hopkins University and complied with the National Institutes of Health Guide for the Use of Experimental Animals to ensure minimal animal use and discomfort.

DALDA was purchased from US Biologicals (USA), and methylnaltrexone bromide and d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) were purchased from Sigma-Aldrich (USA). Other drugs were purchased from Sigma-Aldrich or Tocris Bioscience (United Kingdom). Stock solutions were freshly prepared as instructed by the manufacturer.

L5 spinal nerve ligation (SNL) surgery was used for the induction of neuropathic pain in rats. The procedure was a modification of that described in our previous studies.^19,20 

After rats were anesthetized (2% isoflurane), a small slit was cut in the atlantooccipital membrane, and a 6- to 7-cm piece of saline-filled PE-10 tubing was inserted. We confirmed intrathecal drug delivery by injecting lidocaine (400 μg/20 μl, Hospira, USA), which resulted in a temporary motor paralysis of the lower limbs.^19,20 

Animals were allowed to recover from surgery for 2 weeks before any behavior testing was done. Animals were acclimatized and habituated to the test environment. All procedures have been described in our previous studies.^19–21 

To assess mechanical hypersensitivity to punctuate mechanical stimuli, we measured paw withdrawal threshold (PWT) to von Frey filaments. Each filament (0.38 to 13.1 g) was applied to the test area on the plantar surface of the hind paw for 4 to 6 s according to the up-down method.^21–23 

To test for signs of heat hypersensitivity, we used the Hargreaves test, which measures paw withdrawal latency (PWL) to radiant heat stimuli. Radiant heat was applied to the plantar surface of each hind paw for three times (3- to 5-min interval) with a plantar stimulator analgesia meter (IITC model 390, USA). A cutoff time of 20 s was used to prevent tissue damage.

We used the rota-rod test to assess the well-known motor impairment side effects of opioids. Rats were acclimatized and trained on a rotating rod (Ugo Basile, Italy) that accelerated from 0 to 30 rpm in 180 s. On the day of testing, rat performance on the rod was measured before (predrug baseline) and 45 min after administration of DALDA or morphine. The time (in seconds) that each animal remained on the accelerating rod without falling was recorded.^19,24 

The open field test was used to assess the effect of systemic DALDA administration on spontaneous exploration and locomotor activity of rats. Rats were placed in an open field chamber (73 × 45-cm rectangular plastic box with a wall height of 33 cm) for 10 min. Their behavior was video recorded, and parameters such as total distance travelled; mean travel speed; and number of border periphery, internal periphery, and center crossings were analyzed by SMART 3 software (Panlab Harvard Apparatus, USA).

In anesthetized rats, we performed tracheotomy, mechanical ventilation, and extracellular recordings of single dorsal horn neuronal activity as described in our previous studies.^20,21  Briefly, a laminectomy was performed at vertebral levels T12 to L1 corresponding to lumbar enlargements at spinal segments L3 to S1. During neurophysiologic recording, animals were paralyzed with intraperitoneal pancuronium bromide (0.15 mg/kg, Elkins-Sinn Inc., USA) to facilitate controlled ventilation. Only wide-dynamic range (WDR) neurons with defined receptive fields (RFs) in the plantar region of the hind paw were studied. The cutaneous RFs of WDR neurons were mapped, and a single site (most sensitive site) near the center of the RF was chosen for application of test stimuli. Analog data were collected with a real-time, computer-based data acquisition and processing system (CED Spike 2, United Kingdom). WDR cells were identified by their characteristic responses.^20,21  The evoked responses of WDR neurons to a series of mechanical (brushing, graded von Frey monofilaments: 1 to 15 g, Stoelting Co., USA) and electrical test stimuli (0.1 to 10 mA, 2 ms) were examined. The WDR neuronal response to a suprathreshold electrical stimulus consists of an early A-component (0 to 100 ms) and a later C-component (100 to 500 ms).

Experiments were conducted as we have described previously.^19,25,26  Briefly, dorsal root ganglions (DRGs) from rats were collected in cold DH10 medium and treated with enzyme solution at 37°C. Neurons were loaded with Fura-2-acetomethoxyl ester (Molecular Probes, USA) for 45 min in the dark at room temperature.^25,26  After being washed, cells were imaged at 340 and 380 nm excitation for detection of intracellular free calcium. Calcium imaging assays were performed by an experimenter blind to drug treatment. For isolectin IB4 labeling studies, dissociated DRG neurons were cultured in an incubator at 37°C. After 24 to 48 h, neuron cultures were treated with fluorescein-labeled Griffonia simplicifolia lectin I-isolectin B4 (1:500; Vector Laboratories, USA) for 10 min at room temperature and rinsed for 3 min. Then these cultures were loaded with Fura-2-acetomethoxyl ester. After calcium imaging assays, we selected small-diameter DRG neurons (less than 25 μm) to analyze the changes in calcium concentration ([Ca²⁺]). Cells were divided into two groups: IB4⁻ and IB4⁺ neurons.

To establish the dose–response functions, we normalized PWT and PWL data by calculating maximum possible effect (MPE) values, as PWTs are often at the cutoff values in naive animals. MPE values for inhibiting mechanical hypersensitivity were calculated with the equation: MPE (%) = [1 − (Cutoff PWT − Postdrug PWT)/(Cutoff PWT − Predrug PWT)] × 100, where cutoff PWT = 21.5 g. Because PWLs in naïve animals do not reach the cutoff value (20 s), MPE values for inhibiting heat hypersensitivity were calculated with the equation: MPE (%) = [1 − (Preinjury PWL − Postdrug PWL)/(Preinjury PWL − Predrug PWL)] × 100. Thus, the MPE value for inhibiting heat hyperalgesia may exceed 100%. In electrophysiology studies, we compared the number of action potentials evoked by test stimuli between predrug and postdrug conditions. For analysis of windup, we plotted the C-component of WDR neurons evoked by each stimulus against the stimulation number in a train of 16 stimuli. We compared the A- and C-component produced by graded electrical stimuli and total C-component in response to windup stimulation between predrug and postdrug conditions.

There were no data missing for any of the variables. The methods for statistical comparisons in each study are given in the figures. The number of animals used in each study was based on our experience with similar studies. We randomized animals to the different treatment groups and blinded the experimenter to drug treatment to reduce selection and observation bias. STATISTICA 6.0 software (StatSoft, Inc., USA) was used to conduct all statistical analyses. The Tukey honestly significant difference post hoc test was used to compare specific data points. Bonferroni correction was applied for multiple comparisons. Two-tailed tests were performed; P < 0.05 was considered significant in all tests.

To investigate the therapeutic utility of DALDA on neuropathic pain, we first examined the effects of systemic administration of DALDA on SNL-induced mechanical hypersensitivity. Compared with preinjury baseline, paw withdrawal threshold (PWT) of nerve-injured (ipsilateral) hind paw to mechanical stimuli was significantly decreased at 2 to 3 weeks post-SNL. Subcutaneous administration of DALDA in male SNL rats dose-dependently increased the ipsilateral PWTs at 15, 45, and 120 min (0.2 to 10 mg/kg, n = 6 to 8/dose), compared with the predrug baseline (fig. 1A). The magnitude and duration of DALDA-induced antiallodynic effects increased with dose. We calculated the peak MPE at 45 min postdrug to establish the dose–response function and calculated the dose estimated to produce 50% MPE (ED₅₀) as 4.2 mg/kg. MPEs of 5 and 10 mg/kg doses were significantly higher than the MPE of vehicle (saline, n = 11, fig. 1A). Contralateral PWT did not change significantly after nerve injury or drug treatment (data not shown). Next, to identify the site of action of DALDA, we utilized peripheral and central MOR antagonists. We pretreated nerve-injured male rats with systemic methylnaltrexone (5 mg/kg, intraperitoneally), a peripherally acting MOR-preferring antagonist, 10 min before DALDA treatment (10 mg/kg, subcutaneously, n = 7). Methylnaltrexone, but not saline, completely blocked the antiallodynic effects of systemic DALDA on mechanical hypersensitivity in male SNL rats (fig. 1B, n = 7). In contrast, pretreatment with intrathecal CTOP (0.5 μg/10 μl, n = 7), a highly selective MOR antagonist, did not prevent the antiallodynic effects of systemic DALDA (fig. 1C). The same CTOP pretreatment blocked spinal opioid analgesia induced by intrathecal injection of DALDA (0.5 μg/10 μl, n = 5), suggesting that CTOP effectively blocks MOR activation in the spinal cord. DALDA (10 mg/kg, subcutaneously) also significantly increased the ipsilateral PWTs from predrug baseline in female rats at 2 to 3 weeks post-SNL (n = 6, fig. 1D). There was a trend that peak MPE at 45 min after DALDA treatment in female rats (44.2 ± 13.3%,10 mg/kg, subcutaneously) is lower than that in male rats (75.9 ± 13.8%, n = 7), but the difference did not reach statistical significance (P = 0.11, Student’s t test).

To further examine the effect of systemic DALDA on heat hyperalgesia in nerve-injured rats, we measured PWL with the Hargreaves test before and after drug treatment. At 2 to 3 weeks post-SNL, PWL was significantly decreased in the ipsilateral hind paw (fig. 2A) but not in the contralateral hind paw (fig. 2B). Systemic DALDA dose-dependently attenuated this SNL-induced thermal hyperalgesia in male rats (0.02, 0.1, 0.2, and 2 mg/kg, subcutaneously, n = 8 to 11/group). We used the peak MPEs for systemic DALDA to reverse heat hyperalgesia at 45 min postdrug to establish the dose–response function and calculate ED₅₀ (fig. 2C). The ED₅₀ (0.06 mg/kg) was significantly lower than that required to inhibit mechanical allodynia (4.2 mg/kg) in male SNL rats. MPEs for 0.1, 0.2, and 2 mg/kg doses were all significantly higher than MPE for the vehicle-treated group (saline, n = 11; fig. 2C). An intraperitoneal injection of methylnaltrexone (5 mg/kg), but not saline (n = 9/group), 10 min before DALDA injection completely blocked the antihyperalgesic effect of high-dose DALDA (2 mg/kg, subcutaneously; fig. 2D). Drug treatment did not significantly alter PWL of the contralateral hind paw (fig. 2E). These findings suggest that peripheral MOR activation mediates systemic DALDA-induced inhibition of both mechanical and heat hyperalgesia in nerve-injured male rats. However, inhibition of mechanical hypersensitivity requires a 70-fold greater dose of DALDA than inhibition of heat hyperalgesia. DALDA (10 mg/kg, subcutaneously) also increased the ipsilateral PWLs from predrug baseline in female rats (n = 6, fig. 2F) but did not significantly alter PWL of the contralateral hind paw (fig. 2G).

To further confirm the site of action and delineate the cellular mechanisms involved in DALDA’s modality-preferred inhibition of neuropathic pain, we examined the changes in spinal WDR neuronal response before and after systemic DALDA treatment in male SNL rats. WDR neurons receive converging afferent inputs in both low-threshold A-fibers and high-threshold C-fibers (presumably nociceptive). Yet, the A- and C-fiber–mediated responses to natural stimulation are not readily differentiated in WDR neurons. In contrast, WDR neuronal response to a suprathreshold electrical stimulus (2 ms) can be separated as a short latency A-fiber component (0 to 100 ms) and a longer latency C-fiber component (100 to 500 ms; fig. 3A), based on the calculated conduction velocities. This unique feature of WDR neuronal response allows us to differentiate the drug effects on A- and C-fiber–mediated activities in the same neuron. We then characterized the effects of DALDA on responses of WDR cells to graded intracutaneous electrical stimuli (0.1 to 10 mA, 2 ms) and to windup-inducing stimuli (16 pulses, 0.5 Hz, supra-C-fiber activation threshold, 2 ms) in nerve-injured rats. An electrical search stimulus was applied through a pair of fine needles inserted subcutaneously at the central area of the hind paw. Systemic DALDA (10 mg/kg, intraperitoneally) significantly decreased the C-component, but not the A-component, of WDR neurons to graded intracutaneous electrical stimuli (fig. 3, B–D, n = 13). The stimulus-response functions of C-component (fig. 3D), but not A-component (fig. 3C), were significantly inhibited by DALDA in WDR neurons. We separated A-component into Aβ- (0 to 25 ms) and Aδ-mediated responses (25 to 100 ms) based on the calculated conduction velocities. The total numbers of Aβ- and Aδ-mediated responses to graded electrical stimuli (0.1 to 10 mA) after DALDA treatment (Aβ: 72.1 ± 5.3; Aδ: 29.9 ± 6.3) were not significantly different from predrug baseline (Aβ: 79.2 ± 4.9; Aδ: 29.3 ± 6.8, P > 0.05, paired t test). Saline treatment (n = 10) did not affect WDR neuron response (fig. 3B).

Repetitive electrical stimuli that activate C-fibers may induce temporal summation and transiently enhance the excitability of dorsal horn neurons, a phenomenon called windup (0.5 Hz, fig. 3A). Windup is most prominent in C-fiber–mediated responses of WDR neurons. Total C-component to windup stimuli was significantly decreased at 30 to 45 min after systemic administration of DALDA (10 mg/kg, intraperitoneally; fig. 3B), compared with that at predrug baseline. DALDA also significantly inhibited the windup function (fig. 3E). Saline treatment (n = 10) did not significantly change the A- or C-component of WDR neuronal response to graded electrical stimuli (fig. 3B) or the windup function (data not shown). Importantly, pretreatment with systemic methylnaltrexone (5 mg/kg, intraperitoneally, 15-min pretreatment, n = 9) completely blocked systemic DALDA-induced inhibition of C-component and windup (fig. 3, B–E). These in vivo electrophysiologic findings suggest that systemic administration of DALDA inhibits the C-component of WDR neurons predominantly and the A-component to a much lesser extent in SNL rats. Further, the inhibition of C-component by systemic DALDA likely occurs through the activation of peripheral opioid receptors.

Mechanical hypersensitivity is a characteristic manifestation of neuropathic pain.^27–29  WDR neurons responded to brush stimuli and showed increased firing rates to increasing intensities of punctuate mechanical stimuli (fig. 4, A–C). Although systemic DALDA inhibited mechanical allodynia, which is considered to be mediated by A-fibers, the average responses of WDR neurons to brush stimulation and the stimulus-response functions to graded punctuate mechanical stimuli were not significantly changed after systemic DALDA treatment (10 mg/kg, intraperitoneally, n = 13) compared with predrug baseline (fig. 4B). Yet, individual WDR neurons responded differently to DALDA treatment. Of 13 neurons, 6 showed a total response to graded mechanical stimuli that was decreased to less than 74% of predrug level, which is more than 2 SD less than the mean value after saline treatment (122.2 ± 24.1%, mean ± SD, n = 8; fig. 4D).Yet, two neurons paradoxically showed an increase in response to more than 170% of predrug level. Thus, quantitative mechanical testing showed different patterns of response to systemic DALDA treatment in WDR neurons of SNL rats. Saline treatment did not affect WDR neuronal response to mechanical stimulation (fig. 4C; n = 8).

Recent studies have suggested that activation of TRPV1-expressing and MrgD-expressing DRG neurons is critical to heat and mechanical pain signaling, respectively.^30–33  Therefore, we examined whether the modality preference we observed in our behavioral tests after systemic DALDA administration is paralleled by differential effects on the excitation of these two subpopulations of primary sensory neurons. We performed calcium imaging studies to examine the effects of DALDA on the [Ca²⁺] increase induced by capsaicin, which activates TRPV1⁺ neurons, and β-alanine, which activates MrgD⁺ neurons (fig. 5, A and B). Bath application of capsaicin (0.5 μM) increased [Ca²⁺] in 41% of DRG neurons, and β-alanine (1 mM) increased [Ca²⁺] in 17% of DRG neurons (n = 300; fig. 5C). Only 4% of DRG neurons responded to both capsaicin and β-alanine, suggesting very small colocalization of TRPV1 and MrgD in DRG neurons. After a 10-min washout of the first drug, a second application of capsaicin or β-alanine produced only a slightly smaller increase in [Ca²⁺], suggesting minimal desensitization. However, pretreatment with DALDA (10 min, bath application) dose-dependently blocked the [Ca²⁺] increase to the second application of capsaicin and β-alanine (fig. 5D). Importantly, MPEs for 0.5 and 1 μM DALDA to inhibit the capsaicin-induced [Ca²⁺] increase were significantly higher than those to inhibit the β-alanine–induced increase (fig. 5D). The IC₅₀ of DALDA to inhibit the capsaicin-induced increase in [Ca²⁺] was 0.14 μM, whereas that to inhibit β-alanine–induced activation was 1.33 μM (P < 0.01, Student’s t test). Thus, DALDA preferentially inhibits the increase in [Ca²⁺] evoked by capsaicin in DRG neurons.

TRPV1 is highly expressed in peptidergic DRG neurons, whereas MrgD is found mostly in the nonpeptidergic subpopulation. IB4 is a histochemical marker for nonpeptidergic DRG neurons, which can be labeled with IB4-fluorescein isothiocyanate in culture. Bath application of KCl (30 mM), which strongly depolarizes the cell membrane, induced a robust increase in [Ca²⁺] in both IB4⁺ and IB4⁻ neurons loaded with Fura-2-acetomethoxyl ester (fig. 6, A–C). As a control, sequential treatment with KCl caused only a modest reduction in the [Ca²⁺] increase. Importantly, DALDA (n = 57), but not vehicle (n = 56), significantly inhibited the KCl-evoked [Ca²⁺] increase in IB4⁻ neurons (fig. 6D), which are likely peptidergic cells. However, in IB4⁺ neurons, DALDA (n = 62) was no more effective than vehicle (n = 29).

To further delineate the role of TRPV1⁺ neurons in DALDA-induced amelioration of mechanical and thermal hypersensitivity in nerve-injured rats, we injected adult male rats 7 days post-SNL with resiniferatoxin (0.1 mg/kg, intraperitoneally, n = 9), a highly potent and selective TRPV1 agonist that desensitizes TRPV1 receptor and decreases the excitability of TRPV1-expressing neurons. At 7 to 9 days after resiniferatoxin treatment, heat hypersensitivity was abolished in SNL rats (fig. 7A). Systemic resiniferatoxin, but not vehicle (data not shown), further induced heat analgesia, as indicated by increases in PWLs of both injured and uninjured hind paws to levels greater than the preinjury baseline (fig. 7A). However, resiniferatoxin treatment had no effect on nerve injury–induced mechanical hypersensitivity. Importantly, systemic DALDA (10 mg/kg, subcutaneously) was still able to inhibit mechanical hypersensitivity in resiniferatoxin-treated SNL rats, as indicated by a significant increase in PWT at 15 to 120 min postinjection (fig. 7B, n = 9). These data clearly indicate that systemic DALDA has different cellular targets for inhibiting nerve injury–induced mechanical and thermal hypersensitivity.

Finally, to determine whether systemic DALDA produces well-known opioid-related side effects such as motor incoordination and locomotor impairment, we compared male SNL rats treated with DALDA with those treated with morphine. In the rota-rod test, the fall time of rats treated with systemic DALDA (10 and 20 mg/kg, subcutaneously) was not significantly decreased at 45 min after injection compared with pre-DALDA baseline and that in saline-treated rats (fig. 8, A and B, n = 5 to 8/group). In contrast, rats treated with morphine (10 mg/kg, subcutaneously, n = 8; fig. 8B) showed a significant reduction in fall time. In additionally, in the open field test, total distance traveled (in 10 min; fig. 8, C and D), number of center crossings (fig. 8E), mean travel speed, and number of entries at the border and internal periphery (data not shown) were unaffected at 45 min after DALDA treatment (10 mg/kg, subcutaneously) compared with those at predrug baseline and in the saline-treated group (n = 5 to 8/group). Thus, nerve-injured rats displayed normal activity level, gross locomotion, and exploration habits after systemic DALDA treatment. In contrast, all activities were markedly reduced in morphine-treated rats (10 mg/kg, subcutaneously, n = 5/group) compared with pretreatment baseline and saline- and DALDA-treated groups.

The therapeutic utility of centrally penetrating MOR agonists in neuropathic pain treatment is limited by central adverse effects. In this study, we utilized various behavioral, pharmacologic, in vivo electrophysiologic, and molecular biologic tools to demonstrate the peripherally restricted, modality-preferred antihyperalgesic effects of systemic DALDA. We further delineated potential underlying cellular mechanisms that will help to establish the therapeutic utility of peripherally acting opioids for the treatment of neuropathic pain.

Our in vivo electrophysiologic recordings revealed that DALDA predominantly inhibited C-fiber inputs, which signal thermal and noxious mechanical information, to WDR neurons. These findings complement those of the animal behavioral studies, which also showed that systemic DALDA more effectively inhibited heat hypersensitivity (mediated by C-fibers) than mechanical hypersensitivity (likely mediated by A-fibers). Although the A-component of WDR neurons to electrical simulation was not reduced, a subgroup of WDR neurons showed decreased responses to natural mechanical stimulation after systemic DALDA treatment. The reason for this discrepancy is unclear, but it is possible that systemic DALDA may change A-fiber conduction properties, which can be revealed when WDR neurons receive a prolonged barrage of afferent inputs produced by mechanical stimuli (5 s) but may not be observed with a short (2 ms) high-intensity electrical pulse. Systemic DALDA also inhibited windup of the C-component, reflecting a short-term neuronal sensitization to repetitive noxious inputs that occurs during natural stimulation of C-fibers.^34–36  Thus, systemic DALDA may also ameliorate the neuronal sensitization that underlies development of hyperalgesia by inhibiting peripheral noxious inputs.^37–41 

Although pharmacokinetic data are unavailable for subcutaneous DALDA administration in nerve-injured rats, our findings indicate that DALDA may not appreciably accumulate in CNS after systemic administration. Importantly, systemic DALDA-induced inhibition of both mechanical and heat hyperalgesia was blocked by systemic methylnaltrexon but was unaffected by intrathecal CTOP, which blocks spinal MORs over a prolonged period. Because the activation of MORs on dorsal horn neurons would reduce WDR neuronal excitability and inhibit their responses to both A- and C-fiber inputs, the finding that A-component in WDR neurons was not reduced by DALDA may also imply that systemic DALDA does not activate spinal MORs. Although systemic DALDA may activate MOR in the brain regions to induce pain inhibition, the following findings suggest that DALDA inhibits neuropathic pain primarily through peripheral mechanisms in this study. Activation of MORs in CNS would induce both antihyperalgesia and antinociception. DALDA is highly specific to MOR and is 14-fold more potent than morphine.¹⁶  However, unlike morphine, which often induces antinociception, systemic DALDA normalized heat hyperalgesia without producing antinociception (e.g., PWL above preinjury baseline) in nerve-injured rats. In addition, contralateral PWT and PWL in SNL rats did not increase after systemic DALDA treatment. If DALDA had entered the CNS, it would have induced antinociception. In addition, there is also an absence of any demonstrable CNS-related side effects known to morphine in SNL rats after systemic DALDA treatment.

We further examined the mechanisms for modality-preferred pain inhibition by systemic DALDA in male SNL rats. Different peripheral and central mechanisms are involved in mechanical and heat hypersensitivity. Both animal behavioral and in vivo electrophysiologic findings point to a peripheral site of action for systemic DALDA. Multimodal nociceptors in the peripheral nervous system express a wide array of ion channels and receptors that transduce intense thermal, mechanical, chemical, or cold stimuli into electrical activity.^42,43  Some receptors are segregated into different subsets of DRG neurons, such as the “heat receptor” TRPV1 and the “cold receptor” TRPM8. Mas-related G-protein-coupled receptors (e.g., MrgC, MrgD) are also distributed in a mutually exclusive fashion in DRG neurons.^30,33,44,45  Thus, different subpopulations of primary sensory neurons may contribute to the modality-specific differences in DALDA’s effects on mechanical and thermal hyperalgesia.^30,46  Small-diameter DRG neurons, which are presumably nociceptive, generally can be separated into peptidergic and nonpeptidergic subpopulations. The vanilloid receptor TRPV1 is well known for its role in heat pain signaling. TRPV1 and MOR are more highly expressed and colocalized in peptidergic DRG neurons (likely IB4⁻) than in the nonpeptidergic subpopulation. DALDA shows high binding affinity and is highly selectivity for MORs with a selectivity ratio Ki-δ/Ki-μ of 11,400.^16–18  Therefore, DALDA may preferably inhibit heat-sensing TRPV1⁺ neurons by activating MORs. This preference may partially explain the greater efficacy of systemic DALDA to inhibit heat hypersensitivity than mechanical hypersensitivity. In line with this notion, DALDA significantly inhibited the [Ca²⁺] increase induced by KCl in IB4⁻ neurons but not in IB4⁺ neurons. Further, DALDA induced significantly greater inhibition of the [Ca²⁺] increase evoked by capsaicin, which activates TRPV1, than that evoked by β-alanine, which activates MrgD. TRPV1⁺ neurons play a dominant role in heat nociception and hyperalgesia, whereas MrgD⁺ neurons are important to mechanical pain signaling.^30,31,47  MrgD is expressed mostly in nonpeptidergic neurons and rarely colocalize with TRPV1. Indeed, our results also suggest that only 4% of DRG neurons respond to both capsaicin and β-alanine. Systemic treatment with resiniferatoxin, which selectively decreases the excitability of TRPV1⁺ neurons,^48,49  produced a prolonged reversal of heat hypersensitivity, but not mechanical hypersensitivity, in nerve-injured rats. Interestingly, resiniferatoxin treatment did not affect the attenuation of mechanical allodynia by systemic administration of DALDA. This finding suggests that systemic DALDA ameliorates mechanical and heat hypersensitivity via different cellular targets. Together, these findings suggest a potential cellular mechanism by which DALDA preferentially inhibits heat hypersensitivity.

Finally, to study the safety profile of DALDA, we tested DALDA-treated rats in rota-rod and open field tests. In contrast to morphine, systemic DALDA did not impair motor coordination of rats in the rota-rod test, even at the highest doses tested. In addition, although morphine-treated rats exhibited reductions in distance traveled and travel speed in the open field test, the locomotor function of DALDA-treated rats appeared unchanged from baseline. Additional findings from other investigators suggest that DALDA produces only a transient, minor increase in blood pressure and does not affect maternal respiratory, hemodynamic, or metabolic functions, further suggesting that DALDA has a minimal side-effect profile.⁵⁰  However, DALDA may share other peripheral nervous system side effects (e.g., constipation, vomiting, dry mouth) known to peripherally acting μ-opioids after repetitive and long-term drug treatment. The pharmacokinetic, peripheral nervous system side effects and influences on bowel function of DALDA need to be systematic and carefully investigated in future to fully characterize the pharmacologic properties of this molecule. Our recent study suggested that repeated use of loperamide for alleviating neuropathic mechanical hypersensitivity may lead to the development of tolerance, possibly at peripheral opioid receptors.⁵¹  Because different MOR agonists induce different magnitudes of receptor internalization, desensitization, and tolerance processes, it remains to be tested whether DALDA leads to the development of analgesic tolerance or opioid-induced hyperalgesia.

Systemic DALDA (10 mg/kg, subcutaneously) also alleviated mechanical and heat hypersensitivity in female rats after nerve injury. Intriguingly, there was a trend that MPE for DALDA to inhibit mechanical hypersensitivity in female rats was lower than that in male rats, suggesting possible gender-based differences. Because estrous cycles of female may profoundly affect pain response and drug effects,^52–54  future studies need to characterize gender difference and determine effects of estrous cycles on the efficacy and mechanisms of pain inhibition by DALDA. Such studies will help to fully establish the clinical usefulness of peripherally acting opioids for a therapeutic formulation. In summary, our findings suggest that systemic administration of DALDA attenuates both mechanical and heat hypersensitivity in nerve-injured rats through activation of MORs at peripheral but not at central sites. Further, the efficacy of DALDA to inhibit heat hypersensitivity is greater than that to inhibit mechanical hypersensitivity in male rats. Because it does not affect CNS function, DALDA may pose minimal risk for central dose-limiting adverse effects and have low addiction or abuse potential. Hence, DALDA may represent a promising therapeutic alternative to currently used opioids for the treatment of neuropathic pain.

The authors thank Claire F. Levine, M.S. (Department of Anesthesiology/CCM, Johns Hopkins University, Baltimore, Maryland), for editing the manuscript.

This study was supported by grants from the National Institutes of Health (Bethesda, Maryland): NS26363 (to Dr. Raja) and NS70814 (to Dr. Guan); seed grant from the Johns Hopkins Blaustein Pain Research Fund (Baltimore, Maryland) (to Dr. Guan); and a grant from the National Natural Science Foundation of China (Beijing, China) (81428008; to Dr. Wang). Dr. Dong is a faculty of the Howard Hughes Medical Institute (Baltimore, Maryland). This work was facilitated by the Pain Research Core (Baltimore, Maryland) funded by the Blaustein Fund and the Neurosurgery Pain Research Institute at the Johns Hopkins University (Baltimore, Maryland). Dr. Shechter received funding from Ruth L. Kirschstein National Research Service Award-Sponsoring institution (Johns Hopkins University; Grant no. 2T32GM075774-7).

The authors declare no competing interests.