Share this article on:

R-Duloxetine and N-Methyl Duloxetine as Novel Analgesics Against Experimental Postincisional Pain

Wang, Chi-Fei MD; Russell, Gabriella BFA; Wang, Sho-Ya PhD; Strichartz, Gary R. PhD; Wang, Ging Kuo PhD

doi: 10.1213/ANE.0000000000001086
Anesthetic Pharmacology: Research Report

BACKGROUND: Antidepressant S-duloxetine alleviates intractable pain associated with diabetic peripheral neuropathy and fibromyalgia. It also reduces both acute and persistent pain in various animal models. This study addresses whether the enantiomer, R-duloxetine, and the homolog, N-methyl duloxetine, could act as analgesics and whether they block neuronal Na+ channels.

METHODS: The rat incision plus extension model on the dorsothoracic skin was applied to evoke postoperative mechanoallodynia and hyperalgesia, measured for 5 days postoperatively by local responses to von Frey filaments. R-Duloxetine and N-methyl duloxetine were administered systemically (intraperitoneal) or locally (subcutaneous [SC]) 1 hour before the surgery. The block of Na+ currents in rat neuronal GH3 cells was determined under the whole-cell configuration.

RESULTS: Ipsilateral SC injections (2 mg/0.4 mL) of R-duloxetine and N-methyl duloxetine reduced both postoperative allodynia and hyperalgesia by approximately 89% to 99% in the area under the curve of skin responses next to incision over 5 days. Systemic intraperitoneal injections at a higher dosage (10 mg) had smaller analgesic effects (reduced by approximately 53%–69%), whereas contralateral SC injections (10 mg) were ineffective. Both R-duloxetine and N-methyl duloxetine blocked neuronal Na+ currents, with a higher affinity for the inactivated than the resting states. In addition, both drugs elicited significant use-dependent block of Na+ currents when stimulated at 5 Hz.

CONCLUSIONS: R-Duloxetine and N-methyl duloxetine are highly effective against postoperative pain using the skin incision model, and they elicit both tonic and use-dependent block of neuronal Na+ channels. Our results suggest that R-duloxetine and N-methyl duloxetine are applicable as novel analgesics.

Published ahead of print December 7, 2015

From the *College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Anesthesiology, Perioperative, and Pain Medicine, Brigham & Women’s Hospital, Boston, Massachusetts; and Department of Biological Sciences, SUNY at Albany, Albany, New York.

Accepted for publication September 14, 2015.

Published ahead of print December 7, 2015

Funding: This research was funded by National Institutes of Health grant number GM094152 and CA080153.

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Sho-Ya Wang, PhD, Department of Biological Sciences, SUNY at Albany, 1400 Washington Ave., Albany, NY 12222. Address e-mail to sywang@albany.edu.

Duloxetine (an S-enantiomer drug) is a mixed serotonin-norepinephrine reuptake inhibitor and is widely prescribed for major depressive and anxiety disorders. The drug was originally developed as a serotonin-norepinephrine reuptake inhibitor antidepressant because it is twice as potent as the enantiomer R-duloxetine1 in reducing serotonin uptake. Duloxetine is also an US Food and Drug Administration–approved analgesic used for various pain syndromes, including diabetic peripheral neuropathy and fibromyalgia. The underlying mechanism for duloxetine against these pain syndromes remains unclear, but it may involve multiple central nervous system (CNS) targets.2,3

Duloxetine, by the oral gavage or by the intraperitoneal (IP) route, is effective in reducing pain in several animal models. For example, duloxetine (5–30 mg/kg oral) reversed mechanical allodynia behavior in the L5/L6 spinal nerve ligation model of neuropathic pain.4 Likewise, both mechanical and thermal hyperalgesia from inflammation of hind paw by formalin injection5,6 were significantly attenuated by acute systemic duloxetine (30–100 mg/kg, IP). In the tibial neuroma transposition model, duloxetine attenuated the mechanical allodynia effectively when applied IP at 25 to 50 mg/kg.7

Duloxetine has 3 major CNS targets: (1) serotonin transporter (Ki, 4.6 nM), (2) norepinephrine transporter (Ki, 16 nM), and (3) dopamine transporter (Ki, 370 nM).3 In the past, the antidepressant action was often thought to be the primary mechanism for its analgesic efficacy. This theory was addressed later by “Path Analysis,” and the result showed that duloxetine affected pain directly rather than indirectly through mood improvement.8 This article suggested that the analgesic action of duloxetine is because of its ability to enhance both serotonin and norepinephrine neurotransmission in descending modulatory pain pathways.

In addition to these multiple CNS targets, duloxetine, like the antidepressant amitriptyline and the local anesthetic bupivacaine, blocks voltage-gated Na+ channels.9 Because neuronal Na+ channels are present in both CNS and peripheral nervous systems, such a finding expands the possible analgesic action and locus of duloxetine. Recently, we provided evidence that local subcutaneous (SC) duloxetine was effective for cutaneous antihyperalgesia after rat skin incision.10 In contrast, contralateral SC injections of duloxetine failed to reduce postincisional hyperalgesia or allodynia. This observation is consistent with the notion that duloxetine may also act on peripheral targets.

In this report, we explore whether the enantiomer, R-duloxetine, and the homolog, N-methyl duloxetine, could act as novel analgesics via systemic and SC routes. Furthermore, we explore whether both these drugs are also potent Na+ channel blockers.

Back to Top | Article Outline

METHODS

Drugs

R-Duloxetine and duloxetine were purchased from AK Scientific, Inc., Union City, CA. N-methyl duloxetine was obtained from Waterstone Technology, Carmel, IN. Duloxetine and R-duloxetine contain a secondary amine moiety with a calculated pKa value of 9.2 (SPARC software; www.archemcalc.com). In comparison, N-methyl duloxetine has a tertiary amine moiety with a calculated pKa value of 8.7.

Back to Top | Article Outline

Skin Incision, SC or IP Drug Injections, and Assessment of Postoperative Allodynia and Hyperalgesia

The protocol was approved by the Harvard Medical Area Standing Committee on animals. Male Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). Under anesthesia with sevoflurane inhalation (Abbott Laboratories, North Chicago, IL; 2%–5%), a single, 1-cm longitudinal (and 1-cm parallel to the midline) incision of the shaved skin was made with a sterile surgical blade no. 10, in a sterile field. The SC space was then extended by separation from the underlying muscle by blunt dissection of the fascia for 1 cm wide from the incision, a procedure termed “skin incision plus extension” (SIE).11 The wound was closed with 2 sutures of 3-0 silk (Look®; Surgical Specialties Corp, Reading, PA).

R-Duloxetine and N-methyl duloxetine were dissolved in a saline vehicle solution (0.9% NaCl with 5% Tween-80 and 5% ethanol) for in vivo studies. Each drug was injected SC in a volume of 0.4 mL by a 0.5-mL tuberculin syringe through a 28-G needle (Becton Dickinson and Co., Franklin Lakes, NJ) inserted to its full length. This volume was evenly dispersed to raise a bubble under the skin of approximately 0.8 to 1.2 cm in diameter. One hour after injection, the SIE procedure was made carefully just above the injected area. Studies on intact skin showed that this timing would give maximum block of injected bupivacaine by the time of the incision.10 The IP injection was performed 1 hour before the skin incision in a volume of 1 mL through a 28-G needle with a total of 10 mg drug per rat. Eight rats were used in each cohort unless otherwise indicated. At the time of drug injection, rats weighted approximately 250 to 300 g.

Mechanical stimulation by nylon monofilaments (von Frey hairs [VFHs]), with bending forces of 4 and 15 g, was used to assess postoperative pain, categorized as allodynia and hyperalgesia, respectively. Responsiveness was scored by the number of times that the rat gave a robust contraction of the subdermal muscles under the poked location (the cutaneous trunci muscle reflex) to 6 sequential stimuli with the same VFH. This cutaneous trunci muscle reflex was taken as a pain response. Specifically, each VFH was applied 6 times in any 1 trial done at a specified time, each “poke” of the back skin was spaced 2 to 3 seconds apart, and the 4-g VFH was applied before the 15-g VFH. The fractional responses (n/6) during each trial were recorded for each rat and averaged over the entire population in that test group.

Responsiveness was tested preoperatively (baseline determination) and then at specific times after surgery by the same tester: 1 hour before operation (drug injection), time zero (immediately before operation), 2 or 10 hours after operation, and once a day for 5 days. Adverse effects of R-duloxetine and N-methyl duloxetine were not noticeable after drug injection. The tester was not blinded to the location or the type of drug used.

The elevated responses from local trauma and inflammation were tested on the area of the skin adjacent to the incision (primary hypersensitivity; at 0.5 and 1 cm), and those due to central sensitization were tested on skin much further away, at 2 cm from the incision, including tests on the contralateral side (secondary hyperalgesia). Such contralateral changes are thought to arise from changes in the sensitivity of the spinal cord and brain that result from the injury-induced afferent input, termed “central sensitization.”12,13 Both postoperative allodynia and hyperalgesia using this rat model last over 2 weeks11 and can be transiently reversed by systemic morphine (2.5 mg/kg IP).14

Back to Top | Article Outline

Data Treatment and Statistical Analysis for SIE

Statistical analyses were performed by GraphPad Prism software (GraphPad, San Diego, CA). Data obtained from skin incision were expressed as the number of responses to punctate mechanical stimulation, graphed, and given as means ± SEM. The integrated hypersensitivity after incision was calculated as the area under the curve (AUC) for the response, minus the baseline response, subtracted at each postoperative time point. The areas were determined by the trapezoidal approximation of individual rats using Origin software (Northampton, MA).11 These AUCs (in units of “fractional response-days”) were compared between drug treatments and the control without drug. Results were analyzed by nonparametric Kruskal-Wallis test followed by the Dunn pairwise multiple comparisons test. This AUC approach over various time points was used to see whether analgesic effects induced by drugs diminish over time. P values of <0.01 were considered statistically significant, and P values of <0.05 were viewed as a likely trend.

Back to Top | Article Outline

Culture of GH3 Cells

Rat pituitary GH3 cells were purchased from American Type Culture Collection (Manassas, VA). Cultured GH3 cells were plated on 30-mm culture dishes and maintained at 37°C in a 5% CO2 incubator in DMEM (Life Technologies, Rockville, MD) containing 10% fetal bovine serum (HyClone, Logan, UT), 1% L-glutamine (Life Technologies, Grand Island, NY), and 1% penicillin and streptomycin solution (Sigma, St. Louis, MO).

Back to Top | Article Outline

Electrophysiology, Data Acquisition, and Statistics

Cultured cells were perfused with an extracellular solution containing (in mM) 65 NaCl, 85 choline-Cl, 2 CaCl2, and 10 HEPES (titrated with tetramethylammonium-OH to pH 7.4). The pipette (intracellular) solution consisted of (in mM) 100 NaF, 30 NaCl, 10 EGTA, and 10 HEPES (titrated with cesium-OH to pH 7.2). R-Duloxetine and N-methyl duloxetine were dissolved in dimethyl sulfoxide at 100 mM and stored at 4°C. Final drug concentrations were made by serial dilution.

The whole-cell configuration of the patch-clamp technique15 was used to record Na+ currents at room temperature. Command voltages were elicited with pCLAMP9 software and delivered by Axopatch 200B (Molecular Devices, Inc., Sunnyvale, CA). Cells were held at −140 mV and dialyzed for 15 to 20 minutes before recording.16 Access resistance was approximately 1 MΩ; series resistance compensation of >95% resulted in voltage errors of ≤2 mV at +50 mV. Curve fitting was performed by MicroCal Origin with a Hill equation for concentration-response studies.17 An unpaired Student t test was used to evaluate estimated parameters (mean ± SEM or fitted value ± SE of the fit); P values of <0.01 were considered statistically significant.

Back to Top | Article Outline

RESULTS

Cutaneous Analgesia Induced by SC R-Duloxetine and N-Methyl Duloxetine for Postoperative Pain

For direct side-by-side comparison, we injected SC the same dosage (0.5% in 400 μL; or 2 mg) of R-duloxetine or N-methyl duloxetine under the same SIE experimental conditions that has been used for duloxetine.10Figure 1 shows that local anesthesia by both drugs was evident immediately after the drug injection, and the reduction of allodynia was present at time 0, 10 hours postoperatively, and continued up to 5 days after a single injection. The analgesic effects occurred at all tested areas, including 0.5, 1, and 2 cm from the ipsilateral incision site, as well as at the contralateral site (*P < 0.05; **P < 0.01). However, at the more distant site, the analgesic effects for both drugs appeared to be relatively attenuated. Reduction of allodynia was likewise found at the contralateral side, but more so for N-methyl duloxetine than for R-duloxetine, which wore off 1 day after drug injection (Fig. 1D). Figure 2 shows the early local anesthesia and the reduction of hyperalgesia by both drugs with their antihyperalgesic effects persisting for 5 days. The local analgesia was so profound near the incision site (0.5–1 cm) that the VFH responses were actually lower than the baseline level (Fig. 2, A and B; **P < 0.01) from day 0 to day 3. This below-baseline phenomenon resembles the analgesic effect induced by local bupivacaine-releasing microspheres with a dosage of 40 mg.11

Figure 1

Figure 1

Figure 2

Figure 2

Table 1

Table 1

Table 2

Table 2

To quantify the overall analgesic effect of R-duloxetine and N-methyl duloxetine, we integrated the AUC of Figures 1 and 2 from day 0 to day 5 (Table 1). For comparison, Table 1 also lists the AUC values from the treatment of duloxetine under identical conditions as reported previously.10N-Methyl duloxetine appeared highly effective in reducing allodynia (by approximately 99% or approximately 1% remaining) and hyperalgesia (by approximately 95%) next to the incision site. In general, the AUC values followed the order of N-methyl duloxetine ≤ R-duloxetine ≤ duloxetine ≤ control vehicle. Statistical analyses using the Kruskal-Wallis and Dunn multiple comparison tests indicate that R-duloxetine and N-methyl duloxetine are highly effective with P < 0.05 (*) and P < 0.01 (**) in most of the ipsilateral areas tested compared with the control AUC without drug (Table 2). For tests on the contralateral areas, the reduction in AUC from day 0 to day 5 did not reach statistical significance for R-duloxetine, but it did for N-methyl duloxetine (Table 2). In general, SC N-methyl duloxetine appeared more potent than SC R-duloxetine and duloxetine as analgesics in this SIE pain model, although the differences in AUC data among N-methyl duloxetine, R-duloxetine, and N-methyl duloxetine (Table 1) did not reach statistical significance (not shown in Table 2). In addition, we integrated the cumulative AUC curves from day 0 to various time points (Figs. 1 and 2). These analyses gave us means to evaluate the analgesic efficacy of drugs up to the given time point.

Back to Top | Article Outline

Cutaneous Analgesia Induced by IP and Contralateral SC Injections of R-Duloxetine and N-Methyl Duloxetine for Postoperative Pain

To determine whether systemic applications of R-duloxetine and N-methyl duloxetine are effective in reducing allodynia and hyperalgesia after skin incision, we applied both drugs via IP and contralateral SC injections at a dosage of 10 mg (or 5× greater than 2 mg at the incision site). Figure 3 shows that R-duloxetine and N-methyl duloxetine greatly reduced allodynia immediately after IP injections. However, the analgesic effects of 10 mg N-methyl duloxetine on allodynia lasted only for 2 days (Fig. 3, A–C; ●, [Black up-pointing triangle]). Figure 3 also shows that the contralateral applications of R-duloxetine and N-methyl duloxetine displayed minimal analgesic effects at the skin incision site (Fig. 3, A–C;○,△). Analgesic effects by these contralaterally injected drugs were likewise minimal at the contralateral site (Fig. 3D).

Figure 3

Figure 3

Figure 4A to 4D shows that 10 mg doses of R-duloxetine and N-methyl duloxetine also reduced postoperative hyperalgesia via the IP injections. The antihyperalgesic effects at the SIE site by contralateral injections of R-duloxetine and N-methyl duloxetine were minimal, whereas, at the contralateral site, the analgesic effects were modest but long-lasting (Fig. 4D;○,△).

Figure 4

Figure 4

Table 3

Table 3

Table 4

Table 4

For quantitative analyses, we integrated the AUC over the 5-day time course as measured in Figures 3 and 4, and the results are listed in Table 3. IP delivery of R-duloxetine reduced allodynia (by approximately 69%) and hyperalgesia (by approximately 65%) next to the incision site. The table shows a general trend of AUC values: R-duloxetine (IP) ≤ N-methyl duloxetine (IP) ≤ N-methyl duloxetine (contralateral) ≤ R-duloxetine (contralateral) ≤ control vehicle. Statistical analyses were performed using Kruskal-Wallis and Dunn multiple comparison tests (Table 4). Our results indicate that IP injections of 10 mg R-duloxetine and 10 mg N-methyl duloxetine are quite effective for the reduction of allodynia and hyperalgesia at the SIE side in our pain model compared with those injected with vehicle. However, regarding the reduction in allodynia by N-methyl duloxetine, modest differences in AUC values between drug treatment and control were reached only up to 3 days (Fig. 3A, ●; 0.5 cm away) (*P < 0.05), probably because the drug wore off. Another exception was at the 2.0-cm area for allodynia (4-g VFH) after R-duloxetine IP injection, where the reduction in AUC reached statistical significance only up to day 3 (Fig. 3C, ●) (**P < 0.01). When drugs at a dosage of 10 mg were injected SC at the contralateral side, the analgesic effects on allodynia and hyperalgesia were minimal at the ipsilateral incision side. The reduction in hyperalgesia by R-duloxetine injected at the contralateral side was modest at day 3 but did not reach statistical significance (Fig. 4D, ○; P < 0.05).

Back to Top | Article Outline

Block of Resting and Inactivated Na+ Channels by R-Duloxetine and N-Methyl Duloxetine

To understand how R-duloxetine and N-methyl duloxetine work as analgesics via local injections, we investigated whether these drugs block Na+ channels in neuronal cells. Voltage-gated Na+ currents were generated by a test pulse at +50 mV and recorded before and after the drug treatment. Figure 5A shows that the resting block of neuronal Na+ currents induced by 10 μM R-duloxetine (left panel) and N-methyl duloxetine (right panel) was approximately 10% or less when the cell was held at −140 mV. However, when a conditioning pulse of −70 mV was applied first for 10 seconds, the block increased substantially to approximately 70% or more of Na+ currents (Fig. 5B, left and right panels). Under this 10-second conditioning pulse, drugs interacted primarily with the inactivated Na+ channels.16

Figure 5

Figure 5

The dose-response curves for the block of resting and inactivated Na+ channels at various drug concentrations were constructed as described in the Methods section, and the IC50 and Hill coefficient (in bracket) values of R-duloxetine for the resting and inactivated Na+ channels were estimated at 33.8 ± 1.5 μM (1.51 ± 0.11) and 5.82 ± 0.12 μM (1.51 ± 0.04) (n = 6), respectively (Table 5). The IC50 values of duloxetine for the resting and inactivated Na+ channel were estimated at 30.4 ± 1.24 μM (1.44 ± 0.10) and 4.26 ± 0.19 μM (1.54 ± 0.09) (n = 8), respectively (Table 5). The difference in inactivated Na+ channel block by R-duloxetine and duloxetine might have been modest (P < 0.05) because it did not reach statistical significance. These results demonstrated that there is only modest stereoselectivity (by a ratio of 1.37) of neuronal inactivated Na+ channels toward duloxetine (S-form) and R-duloxetine.

Table 5

Table 5

Table 5 also includes IC50 values of 138 ± 15 (1.00 ± 0.06) and 2.80 ± 0.06 (1.08 ± 0.02), respectively, for resting and inactivated Na+ channel block by N-methyl duloxetine (n = 6). Under the same conditions, bupivacaine displayed IC50 values of 189.6 ± 22.3 and 9.6 ± 0.9 μM, respectively, for resting and inactivated Na+ channels in GH3 cells, whereas amitriptyline showed 39.8 ± 2.7 and 0.9 ± 0.1 μM, respectively.18 Therefore, the IC50 values for inactivated Na+ channels in GH3 cells follow the order of amitriptyline (0.9 μM) < N-methyl duloxetine (2.80 μM) < S-duloxetine (4.26 μM) < R-duloxetine (5.82 μM) < bupivacaine (9.6 μM).

Back to Top | Article Outline

Use-Dependent Block of Neuronal Na+ Currents by R-Duloxetine and N-Methyl Duloxetine

Figure 6

Figure 6

To determine whether R-duloxetine and N-methyl duloxetine elicit use-dependent block of Na+ currents, we applied repetitive pulses of +50 mV for 20 milliseconds at 5 Hz. Figure 6A and 6B shows the superimposed Na+ currents before and after the 10 μM treatments of R-duloxetine and N-methyl duloxetine, respectively. The use-dependent block reached approximately 75% for R-duloxetine in peak current reduction (pulse 1 versus pulse 60), approximately 72% for duloxetine, and approximately 45% for N-methyl duloxetine (Table 5). The magnitude of the use-dependent block by these 3 drugs is much higher than the control value without drug (approximately 4%; P < 0.01).

Back to Top | Article Outline

DISCUSSION

Our in vivo experiments using the rat skin incision pain model show that R-duloxetine and N-methyl duloxetine are highly effective in reducing postoperative hyperalgesia and allodynia, both via the IP and the SC routes. The in vitro studies using GH3 cells show that these drugs are potent neuronal Na+ channel blockers and that they interact strongly with the inactivated states of neuronal Na+ channels. Our data thus indicate that (1) the enantiomer, R-duloxetine, and the homolog, N-methyl duloxetine, are applicable as novel analgesics for pain relief, and that (2) like duloxetine, these drugs probably act via multiple pathways, including the inhibition of sodium channels,10 as well as the inhibition of serotonin and norepinephrine reuptake.1

Back to Top | Article Outline

R-Duloxetine and N-Methyl Duloxetine as Analgesics for the Reduction of Postoperative Pain

When applied SC, both R-duloxetine and N-methyl duloxetine at a dosage of 2 mg are highly effective in the reduction of allodynia (Fig. 1) and hyperalgesia (Fig. 2) for several days. Interestingly, the duration of analgesia induced by these drugs is also surprisingly comparable with that induced by local, slow bupivacaine-releasing microspheres at a 20-time dosage of 40 mg.11 On the basis of the AUC data (Table 1), the reduction of allodynia and hyperalgesia from the control level mostly follows the order of N-methyl duloxetine ≥ R-duloxetine ≥ duloxetine. We hypothesize that this efficacy in pain relief is because of the local action of SC R-duloxetine and N-methyl duloxetine. This hypothesis is supported by the fact that the injection of these drugs at the contralateral site with a dosage as high as 10 mg did not produce comparable analgesic effects at the skin incision site (Figs. 3 and 4). Such an observation contradicts the notion that antimechanohypersensitivity induced by these drugs, as injected SC, is through a systemic route. Paradoxically, systemic IP applications of R-duloxetine and N-methyl duloxetine at a dosage of 10 mg are very effective in the reduction of postoperative pain (Figs. 3 and 4). Why then did the 10 mg of these drugs injected at contralateral site fail to reduce postoperative pain at the skin incision site? This phenomenon could be explained by the fact that the plasma concentration of local anesthetics during local anesthesia is highly dependent on the route of injection.19 Accordingly, we envision that the uptake of R-duloxetine and N-methyl duloxetine by SC capillaries is relatively slow at the contralateral skin incision site and that the drug in the blood stream does not reach its therapeutic threshold. In contrast, the drug uptake by capillaries via the IP route is likely more rapid.

Back to Top | Article Outline

Central Modes of Actions by R-Duloxetine and N-Methyl Duloxetine

Perahia et al.2 suggested that duloxetine exerts direct analgesic effects by virtue of its relatively balanced mode of inhibitory actions on serotonin and norepinephrine reuptake in patients with major depressive disorder, diabetic peripheral neuropathic pain, and fibromyalgia syndrome. They inferred that these analgesic effects are independent of, and in addition to, the antidepressant effects and that duloxetine targets the descending inhibitory spinal pain pathways in the CNS for its efficacy in the treatment of chronic pain. A parallel inference of direct pain relief by duloxetine was reached by Goldstein et al.8 using path analysis on a 24-hour average pain score in patients with painful diabetic neuropathy. We have found no report in the literature to suggest that N-methyl duloxetine is effective against serotonin or norepinephrine reuptake in the CNS, but it certainly shows promise as an analgesic, as shown in this report.

After IP injections, we should bear in mind a possible local contribution of these drugs because the skin area can be likewise reached by drug via the blood vessels. Because the IP dosage is 5-fold higher than the ipsilateral SC dosage (10 vs 2 mg) for eliciting comparable analgesic effects, R-duloxetine and N-methyl duloxetine may, in theory, arrive at the skin incision site via capillaries. In the remaining text, we will discuss additional modes of actions by R-duloxetine and N-methyl duloxetine.

Back to Top | Article Outline

Block of Neuronal Voltage-Gated Na+ Channels by R-Duloxetine and N-Methyl Duloxetine in a State-Dependent Manner

Many antidepressants block voltage-gated Na+ channels.20 For example, amitriptyline inhibits Na+ currents, and it preferentially binds with the inactivated states with high affinities via the local anesthetic receptor site within the Na+ channel.21 This mode of action by amitriptyline suggests that such an antidepressant could inhibit synaptic transmission in CNS by damping neuronal action potentials and reducing evoked release of both excitatory and inhibitory transmitters. Interestingly, R-duloxetine and N-methyl duloxetine also block the inactivated Na+ channels in GH3 cells and may act as an analgesic in a manner similar to tricyclic amitriptyline and antidepressant duloxetine. We chose to study GH3 cells because they express Na+ channel Nav1.1, Nav1.2, Nav1.3, and Nav1.6 isoforms22; among which Nav1.2 and Nav1.6 are also found in peripheral nerves.

There is no evidence to support a strong stereoselectivity of inactivated Na+ channels toward duloxetine and R-duloxetine (Table 5; 4.27 vs 5.82 μM, respectively). With respect to the block of Na+ currents, both amitriptyline and duloxetine seem to interact more strongly with the open state than with the inactivated state.9,21 Likewise, the open- and inactivated-channel block of neuronal Nav1.7 Na+ channels displayed IC50 value of 0.25 and 1.79 μM, respectively. Nav1.7 Na+ channels are expressed only in the peripheral nervous systems, and they are recognized as potential targets for pain therapeutics.23 Importantly, Nav1.7-knockout mice show a mechanical deficit in the hairy skin but not in the paw.24 For cardiac hNav1.5 inactivation-deficient Na+ channels, we found that the IC50 values displayed a ranking order of R-duloxetine (0.53 ± 0.01 μM) < duloxetine (0.77 ± 0.02 μM) < N-methyl duloxetine (1.34 ± 0.04 μM) (n = 8).

Like duloxetine, R-duloxetine and N-methyl duloxetine elicit significant use-dependent block of neuronal Na+ currents in GH3 cells (Table 5; Fig. 6). This use-dependent phenomenon may be explained if a specific drug preferentially blocks open Na+ channels.9,25 It has become evident that small ramp current and/or persistent late Na+ currents can cause repetitive impulse firing that underlies chronic or neuropathic pain, as found in patients with painful congenital myotonia, familial primary erythermalgia, or paroxysmal extreme pain disorder.26,27 Thus, open-channel selective drugs that target the window Na+ currents or the persistent late Na+ currents could have significant therapeutic effect on intractable pain.

Back to Top | Article Outline

Other Potential Peripheral Targets for R-Duloxetine and N-Methyl Duloxetine?

In addition to voltage-gated Na+ channels, are there other possible peripheral targets in pain pathways28 that could account for the local analgesic action of R-duloxetine and N-methyl duloxetine? Acute nociceptive, inflammatory, and neuropathic pain are known to originate from the activation of peripheral afferent neurons. In particular, peripheral nerve endings contain various receptors, including those for opioid, α-adrenergic, cholinergic, adenosine, serotonin, capsaicin, and cannabinoid.28 Further studies are needed to address whether these peripheral pain pathways could be modulated by R-duloxetine, N-methyl duloxetine, and duloxetine.

In summary, R-duloxetine and N-methyl duloxetine could act as novel analgesics via both IP and SC routes in a manner similar to duloxetine using the rat skin incision pain model. These 3 drugs may be viewed as multifunctional compounds29 that could be beneficial for multifactorial disorders, such as intractable chronic pain.

Back to Top | Article Outline

DISCLOSURES

Name: Chi-Fei Wang, MD.

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

Attestation: Chi-Fei Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Gabriella Russell, BFA.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Gabriella Russell has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Sho-Ya Wang, PhD.

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

Attestation: Sho-Ya Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Gary R. Strichartz, PhD.

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

Attestation: Gary R. Strichartz has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Ging Kuo Wang, PhD.

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

Attestation: Ging Kuo Wang has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

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

Back to Top | Article Outline

ACKNOWLEDGMENTS

We thank Dr. Chuan-Chin Huang for proofreading the statistical analyses.

Back to Top | Article Outline

REFERENCES

1. Wong DT, Bymaster FP, Mayle DA, Reid LR, Krushinski JH, Robertson DW. LY248686, a new inhibitor of serotonin and norepinephrine uptake. Neuropsychopharmacology. 1993;8:23–33
2. Perahia DG, Pritchett YL, Desaiah D, Raskin J. Efficacy of duloxetine in painful symptoms: an analgesic or antidepressant effect? Int Clin Psychopharmacol. 2006;21:311–7
3. Morphy R, Rankovic Z. Designed multiple ligands. An emerging drug discovery paradigm. J Med Chem. 2005;48:6523–43
4. Iyengar S, Webster AA, Hemrick-Luecke SK, Xu JY, Simmons RM. Efficacy of duloxetine, a potent and balanced serotonin-norepinephrine reuptake inhibitor in persistent pain models in rats. J Pharmacol Exp Ther. 2004;311:576–84
5. Bomholt SF, Mikkelsen JD, Blackburn-Munro G. Antinociceptive effects of the antidepressants amitriptyline, duloxetine, mirtazapine and citalopram in animal models of acute, persistent and neuropathic pain. Neuropharmacology. 2005;48:252–63
6. Munro G. Dopamine D(1) and D(2) receptor agonism enhances antinociception mediated by the serotonin and noradrenaline reuptake inhibitor duloxetine in the rat formalin test. Eur J Pharmacol. 2007;575:66–74
7. Miyazaki R, Yamamoto T. The efficacy of morphine, pregabalin, gabapentin, and duloxetine on mechanical allodynia is different from that on neuroma pain in the rat neuropathic pain model. Anesth Analg. 2012;115:182–8
8. Goldstein DJ, Lu Y, Detke MJ, Lee TC, Iyengar S. Duloxetine vs. placebo in patients with painful diabetic neuropathy. Pain. 2005;116:109–18
9. Wang SY, Calderon J, Kuo Wang G. Block of neuronal Na+ channels by antidepressant duloxetine in a state-dependent manner. Anesthesiology. 2010;113:655–65
10. Wang CF, Russell G, Strichartz GR, Wang GK. The local and systemic actions of duloxetine in allodynia and hyperalgesia using a rat skin incision pain model. Anesth Analg. 2015;121:532–44
11. Ohri R, Wang JC, Blaskovich PD, Pham LN, Costa DS, Nichols GA, Hildebrand WP, Scarborough NL, Herman CJ, Strichartz GR. Inhibition by local bupivacaine-releasing microspheres of acute postoperative pain from hairy skin incision. Anesth Analg. 2013;117:717–30
12. Brennan TJ, Vandermeulen EP, Gebhart GF. Characterization of a rat model of incisional pain. Pain. 1996;64:493–501
13. Zahn PK, Brennan TJ. Incision-induced changes in receptive field properties of rat dorsal horn neurons. Anesthesiology. 1999;91:772–85
14. Duarte AM, Pospisilova E, Reilly E, Mujenda F, Hamaya Y, Strichartz GR. Reduction of postincisional allodynia by subcutaneous bupivacaine: findings with a new model in the hairy skin of the rat. Anesthesiology. 2005;103:113–25
15. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85–100
16. Wright SN, Wang SY, Kallen RG, Wang GK. Differences in steady-state inactivation between Na channel isoforms affect local anesthetic binding affinity. Biophys J. 1997;73:779–88
17. Cota G, Armstrong CM. Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent. J Gen Physiol. 1989;94:213–32
18. Gerner P, Mujtaba M, Sinnott CJ, Wang GK. Amitriptyline versus bupivacaine in rat sciatic nerve blockade. Anesthesiology. 2001;94:661–7
19. Covino BG, Vassallo HG Pharmacokinetic Aspects of Local Anesthetic Agents, Local Anesthetics: Mechanism of Action and Clinical Use. 1976 New York Grune & Stratton, Inc:95–122
20. Dick IE, Brochu RM, Purohit Y, Kaczorowski GJ, Martin WJ, Priest BT. Sodium channel blockade may contribute to the analgesic efficacy of antidepressants. J Pain. 2007;8:315–24
21. Wang GK, Russell C, Wang SY. State-dependent block of voltage-gated Na+ channels by amitriptyline via the local anesthetic receptor and its implication for neuropathic pain. Pain. 2004;110:166–74
22. Vega AV, Espinosa JL, López-Domínguez AM, López-Santiago LF, Navarrete A, Cota G. L-type calcium channel activation up-regulates the mRNAs for two different sodium channel alpha subunits (Nav1.2 and Nav1.3) in rat pituitary GH3 cells. Brain Res Mol Brain Res. 2003;116:115–25
23. Levinson SR, Luo S, Henry MA. The role of sodium channels in chronic pain. Muscle Nerve. 2012;46:155–65
24. Minett MS, Eijkelkamp N, Wood JN. Significant determinants of mouse pain behaviour. PLoS One. 2014;9:e104458
25. Wang GK, Calderon J, Wang SY. State- and use-dependent block of muscle Nav1.4 and neuronal Nav1.7 voltage-gated Na+ channel isoforms by ranolazine. Mol Pharmacol. 2008;73:940–8
26. George AL Jr. Inherited disorders of voltage-gated sodium channels. J Clin Invest. 2005;115:1990–9
27. Dib-Hajj SD, Cummins TR, Black JA, Waxman SG. Sodium channels in normal and pathological pain. Annu Rev Neurosci. 2010;33:325–47
28. Sawynok J. Topical and peripherally acting analgesics. Pharmacol Rev. 2003;55:1–20
29. Bansal Y, Silakari O. Multifunctional compounds: smart molecules for multifactorial diseases. Eur J Med Chem. 2014;76:31–42
© 2016 International Anesthesia Research Society