bs_bs_banner
Pain Medicine 2014; 15: 1771–1780 Wiley Periodicals, Inc.
NEUROPATHIC PAIN SECTION Original Research Article Ranolazine Attenuates Mechanical Allodynia Associated with Demyelination Injury
Departments of *Physical Medicine and Rehabilitation, † Neurology, ††Pharmacology and Experimental Therapeutics, ‡Pain Mastery Center of Louisiana, Louisiana State University Health Sciences Center, New Orleans, Louisiana; §Department of Psychology, Nicholls State University, Thibodaux, Louisiana; ¶ Gilead Sciences Inc., Palo Alto, California; **Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky, USA Reprint requests to: Harry J. Gould, III, MD, PhD, Department of Neurology, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA. Tel: 504-568-4080; Fax: 504-568-7130; E-mail: [email protected]. Disclosure: ID is a senior advisor at Gilead Sciences Inc., producer of ranolazine.
of this drug in a newly validated model of demyelination injury that responds uniquely to a number of treatment options. Methods. After determination of baseline nerve conduction velocities (NCVs) and withdrawal responses from heat and mechanical stimulation in male Sprague-Dawley rats (300–350 g), 1 μg/30 μL of doxorubicin was injected into one sciatic nerve. The contralateral nerve provided a sham-injected control. Two weeks after doxorubicin injection, NCV and sensitivity to heat and mechanical stimulation were reassessed before and after treatment with ranolazine (10, 30, 50 mg/kg) administered intraperitoneally using an experimenter-blinded, randomized design. Results. Doxorubicin injection produced a significant hyperalgesic effect in response to mechanical but not heat stimulation. Conduction velocities in the injected limbs were reduced when compared with controls. Ranolazine reduced mechanical allodynia with peak efficacy at 30 mg/kg. Fifty milligram/kilogram ranolazine restored NCVs by approximately 50%, but had no effect in the uninjected limb.
Abstract
Conclusions. Ranolazine exerts broad-spectrum actions to reduce mechanical allodynia that is associated with peripheral demyelination injury.
Objective. The aim of this study was to determine whether ranolazine, a new medication that targets sodium channels to improve cardiac ischemia and angina, could be an effective analgesic agent for pain associated with demyelination injury.
Key Words. Ranolazine; Allodynia
Background. Many agents have been used to treat neuropathic pain but not all neuropathic conditions respond similarly to treatment. We have demonstrated that ranolazine, an agent that blocks voltagegated sodium channels Nav 1.4, 1.5, 1.7, and 1.8, is effective in attenuating mechanical hyperalgesia in both complete Freund’s adjuvant and spared nerve injury preclinical models of inflammatory and neuropathic pain, respectively. Here we test the efficacy
Doxorubicin;
Pain;
Introduction Sodium channels are responsible for the generation and propagation of electrochemical excitability and thus have a significant influence on the function of excitable cells [1]. Injury changes the expression of sodium channels in peripheral nerves. These changes are well correlated with the perception and reporting of pain [2–11], but the patterns of hypersensitivity associated with particular disease 1771
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
Harry J. Gould, III, MD, PhD,*,†,‡ R. Denis Soignier, PhD,‡,§ Sung R. Cho, MD,*,‡ Christian Hernandez, MD,†,‡ Ivan Diamond, MD, PhD,¶ Bradley K. Taylor, PhD,** and Dennis Paul, PhD†,‡,††
Gould et al. states and their response to treatment vary significantly both in preclinical and clinical settings [12–15]. These differences confound our attempts to provide consistent care for patients with pain. Because of the consistent association between hyperalgesia and alterations in sodium channel distribution and density [16], sodium channel blockers are frequently used to produce analgesia for conditions arising from a number of disease states and mechanisms of injury.
Methods Animals All experiments were conducted in accordance with protocols that were approved and monitored by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN, USA) weighing between 300 and 350 g were housed, one animal to a cage, and maintained at 25°C and 60% humidity, on a 12-hour light/dark cycle and allowed access to food and water ad libitum. For 1 week, rats were allowed to acclimate to their surroundings and for 1 hour/day to the testing apparatus. 1772
To determine baseline thresholds to heat stimulation, rats from groups of nine were placed in Plexiglas chambers on a glass plate and were allowed free range of activity within the chamber. The glabrous surface of each hind paw was stimulated sequentially through the glass plate using a halogen light source [27–29]. The latency of paw withdrawal from the onset of stimulation was measured using an IITC analgesiometer (IITC Life Science, Inc., Woodland Hills, CA, USA). The stimulus was automatically discontinued after 10.7 seconds to avoid tissue damage. Each hind paw was stimulated four times during each testing session. The interval between stimulation of paws in a given rat was approximately 2 minutes and no less than 1 minute between trials.
Behavioral Assessment of Mechanical Sensitivity Approximately 30 minutes after heat testing, thresholds to withdrawal from mechanical stimulation were assessed using an IITC Model 2290 electro-von Frey (EVF) anesthesiometer (IITC Life Science, Inc.) [30–32]. For this, the rats were loosely restrained in a cotton towel and allowed to accommodate to the restriction. The tip of the stimulating wand was then applied perpendicular to the skin at four sites on the dorsal surface of each hind paw. The force applied to the paw at the time of paw withdrawal was recorded. The average force applied to the four sites was entered as the subject’s response threshold for the interval and used in all further calculations. A ceiling of 250 g of force was imposed to prevent tissue injury from EVF testing.
CMAPs Finally, after establishing baseline withdrawal thresholds from heat and mechanical stimulation, baseline CMAPs were ascertained in both hind extremities using a NeuroMax1000 unit (Excel Tech, Ltd., Oakville, ON, Canada) to calculate sciatic nerve conduction velocity (NCV). Twenty-seven-gauge, 1.5-cm subdermal needle recording electrodes (CareFusion Corporation, San Diego, CA, USA) were inserted between third and fourth digits of the hind paw. Stimulation of variable duration and voltage to elicit optimal responses was applied distally at the calcaneal tendon and proximally just inferior to the hip joint. The ground electrode was placed between the stimulating and recording sites, and the distance between the stimulating and recording electrodes was measured. The CMAP amplitude and duration and the latency between the stimulus artifact and the initial deflection from the baseline trace were measured and the NCV was calculated. Conduction block was inferred by recording a greater than 10 m/second or 30% decrease in the conduction velocity of the sciatic nerve from baseline values. Due to significant variation in CMAP amplitudes, only NCV criteria were used to confirm demyelination during baseline recording.
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
One such agent is ranolazine (Ranexa®, Gilead Science Inc., Palo Alto, CA, USA), which has been approved by the U.S. Food and Drug Administration (FDA) for treating cardiac ischemia and anginal chest pain [17,18]. The antianginal effects of ranolazine are produced by blocking the late sodium current associated with the voltage-gated sodium channel Nav 1.5. In addition, ranolazine use dependently inhibits Nav 1.7 and 1.8, sodium channels that are linked to the regulation of inflammatory and/or neuropathic pain [19–21]. Because of its affinity for specific sodium channels, we previously tested ranolazine in both the complete Freund’s adjuvant (CFA) [22] and spared nerve injury (SNI) [23] preclinical models of inflammatory and neuropathic pain, respectively. We demonstrated that ranolazine attenuated mechanical and cold hypersensitivity [23], but did not reduce hypersensitivity associated with heat [22]. Because of the consistent analgesic effect on mechanical hyperalgesia present in both inflammatory and neuropathic conditions, we hypothesized that ranolazine would attenuate the mechanical allodynia produced by intraneural (i.n.) injection of doxorubicin, a DNA-intercalating agent that causes delayed subacute, focal, and reversible demyelination by killing Schwann cells and sparing axons [24], analogous to that seen in Guillain–Barré syndrome and chronic inflammatory demyelinating polyneuropathy. This newly validated model of demyelination injury alters the normal distribution and density of sodium channels in the area of the injection site, thereby compromising salutatory conduction of action potentials and resulting in conduction slowing, a common feature of demyelination injury, a reduction in the compound motor action potential (CMAP) [24,25], and a disruption of sensory processing giving rise to an increase in sensitivity to mechanical but not heat stimulation [12,26].
Behavioral Assessment of Heat Hypersensitivity
Analgesic Effects of Ranolazine Treatment Model of Demyelination Injury
Results
Once all the baseline determinations were made, small incisions were made bilaterally at the level of the sciatic notch to expose the sciatic nerves. One sciatic nerve received a 1 μg/30 μL i.n. injection of doxorubicin (Adriamycin®, Sigma-Aldrich, St. Louis, MO, USA). The contralateral nerve was injected with an equal volume of normal saline and served as a sham control. The electrodiagnostician was blinded to which nerve received doxorubicin.
A single injection of doxorubicin into the main trunk of a sciatic nerve produces electrodiagnostic evidence of conduction slowing within 2 weeks of the injection (Figures 1 and 2). The average conduction velocity in the doxorubicin-injected limb was significantly reduced when compared with the contralateral saline-injected controls, 27.79 m/second vs 39.2 m/second, respectively; ANOVA revealed significant interaction: F(2,24) = 3.99, P > 0.05.
Experimental Design
Behavioral Test of Ataxia To determine whether ranolazine nonspecifically reduced central nervous system function such as motor control, we tested its effects in the rotarod test. As previously described [22,23], the ability of animals to remain on an accelerating rotarod was assessed before and after i.p. administration of vehicle or ranolazine (up to 100 mg/kg). Rats were placed on the rotarod, set at an initial rotating speed of 4 rpm. Immediately thereafter, the speed of the rod increased at a rate of 0.5 rpm every 5 seconds to a maximum of 40 rpm. Each rat was trained in this procedure three to nine times until latency to fall was approximately 180 seconds. Triplicate measurements were taken before and then 30, 60, 90, 120, 150, and 180 minutes after ranolazine injection. Statistics The behavioral data were analyzed using GraphPad Prism for Windows® (Version 5.1, GraphPad Software, Inc., San Diego, CA, USA). Sensory data were analyzed using separate one-way analysis of variance (ANOVA) and conduction velocity data were analyzed using a 2 × 3, mixed design ANOVA to determine statistical significance between experimental conditions. The doxorubicin injection resulted in significant reduction in withdrawal threshold to mechanical stimuli (P < 0.01), but not in withdrawal latency from heat stimuli.
Figure 1 The paired traces depict compound motor action potentials (CMAPs) recorded from rats before (A) and 14 days after (B) the intraneural injection of doxorubicin. The conduction velocity (CV) of the sciatic nerve measured 33.9+/− 2.29 m/second at baseline and then 27.79+/− 2.25 m/second following doxorubicin injection. The greater than 30% decrease in CV is evidence of conduction slowing associated with acute demyelination injury. The paired traces recorded from post-doxorubicin injected sciatic nerves after intraperitoneal (i.p.) administration of 30 mg/kg dosing of ranolazine (C) reveal a CV of 39.4+/− 0.69 m/second, essentially returning CV to baseline levels. NCV = nerve conduction velocity. 1773
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
One to 2 weeks after doxorubicin injection, CMAP determinations were repeated and NCVs were calculated. A greater than 30% reduction in NCVs in the doxorubicin injected limb, 27+/− 2 m/second, when compared with that observed in the contralateral control, 40+/− 3 m/second, provided evidence of successful demyelination. Upon substantiation of demyelination, post-doxorubicin sensitivity to heat and mechanical stimulation was then reassessed on two successive days. On the following day, groups of rats received ranolazine dissolved in 0.9% isotonic saline by intraperitoneal (i.p.) injection (0, 10, 30, and 50 mg/kg) or equal volumes of vehicle in a randomized, experimenter-blinded design. Withdrawal thresholds to heat and mechanical stimulation were reassessed 30 minutes after dosing. Post-ranolazine CMAP determinations were also made after behavioral testing was completed.
Gould et al.
Planned comparison of the effects of doxorubicin injection for each time point using the Bonferroni adjustment was carried out. The analysis revealed that the difference between injured and uninjured paws (Figure 2, gray vs black bars) was only significant at the post-injury time point (P < 0.01). No other significant differences were observed. Neither ranolazine nor doxorubicin injection of the contralateral sciatic nerve significantly changed conduction velocity in the control limb. The amplitudes of the CMAPs were fairly consistent but variable enough in both limbs to preclude determination of the presence or extent of axonal injury that resulted from the doxorubicin injection. Variations in conduction velocity, likely due to variability in estimating the position of the electrode tips when measuring the distance between the stimulating and recording electrodes, were not statistically significant. Consistent with the validation studies of this model [12], the i.n. injection of doxorubicin increased sensitivity to mechanical (F [5,70] = 5.56, P < 0.05), but not heat (F[5,66] = 4.67, P = 0.51) stimulation of the hind paw ipsilateral to the injection. Intraneural doxorubicin did not change the response to heat stimulation applied to the ipsilateral or contralateral paw (Figure 3A). By contrast, Figure 3 illustrates that intraneural (i.n.) doxorubicin increased mechanical sensitivity by approximately 30% as 1774
Figure 3 Left side of graphs depicts the sensitivity to heat (A) and mechanical (B) stimulation before (diagonally lined bars) and 14 days after (crosshatched bars) i.n. doxorubicin (dox) injection. The doxorubicin injection resulted in significant reduction in withdrawal threshold to mechanical stimuli (**P < 0.01), but not in withdrawal latency from heat stimuli. Right side of the graphs indicates the dose– response relationship for 0, 10, 30, and 50 mg/kg intraperitoneal (i.p.) doses of ranolazine. At both 30 and 50 mg/kg, withdrawal thresholds to mechanical stimuli were significantly higher vs postinjection baseline (* = P < 0.05). The maximum reduction of mechanical sensitivity observed following ranolazine administration was achieved at the 30 mg/kg dose but did not return to baseline levels. No additional benefit was observed at higher doses. No significant effects of ranolazine were observed on withdrawal latencies from heat stimuli (A) or on contralateral paw sensitivity (data not shown). i.n. = intraneural.
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
Figure 2 The graphs compare the conduction velocities (CVs) of the injected (gray bars) vs contralateral uninjected control (black bars) sciatic nerves recorded from rats prior to doxorubicin injection (baseline) and 14 days after injection both before (post-dox) and after ranolazine treatment (ranolazine) (asterisk denotes significance between black and gray bars at a given time point, P < 0.01). The CV that decreased by approximately 30% of baseline following doxorubicin injection was restored to baseline after 30 mg/kg dosing of ranolazine. Ranolazine had no significant effect on CV in the control nerves.
Analgesic Effects of Ranolazine Treatment allodynia in painful conditions regardless of the mechanism of injury [12,33–35]. Further investigation of ranolazine as an analgesic adjuvant treatment option for this purpose is warranted.
The i.p. administration of ranolazine (0, 10, 30, and 50 mg/kg), but not vehicle, dose-dependently reduced mechanical allodynia produced by doxorubicin injection (ED50 = 8.14 mg/kg ± 3.1; P < 0.001). Ranolazine reduced allodynia by approximately 60% at the maximally effective dose of 30 mg/kg, which does not impair motor control (Figure 4 and see Casey et al. and Gould et al. [22,23]). At higher doses of 50 mg/kg, ranolazine restored NCVs by approximately 50%. Post hoc statistical comparisons made using Dunnett’s test found significant differences between the doxorubicin-injected paws prior to ranolazine (Figure 3, cross-hatched bars) and the shamtreated baseline (P < 0.01) as well as both 30 mg and 50 mg doses of ranolazine (P < 0.05).
Ranolazine is an FDA-approved medication that targets late sodium currents, thereby improving cardiac perfusion and reducing angina [17,18,36,37]. Ranolazine has a modulatory effect on sodium channel subtypes Nav 1.4, 1.5, 1.7, and 1.8 [38–41] and reduces myocardial injury by decreasing the entry of sodium into cardiac myocytes and by reducing the subsequent influx of calcium via the Ca2+/ Na+ exchanger that occurs during ischemia and early reperfusion [19,20,38,42–45]. Because ranolazine targets the Nav 1.7 and 1.8 isoforms of voltage-gated sodium channels that play a significant role in CFA- and SNIinduced mechanical allodynia, the mechanism for the observed analgesic effect seems intuitive [8,22,23,28]. Inflammation is associated with increased expression of Nav 1.3, 1.7, 1.8, and 1.9 isoforms [46]. For example, subcutaneous injection of CFA produces a rapid and dramatic upregulation of the Nav 1.7 isoform of the voltagegated sodium channel in both the large and small neuronal populations in the dorsal root ganglia that innervate the injection site [6,8,28], as well as in the peripheral nerve endings of primary afferents [47,48]. The increase in Nav 1.7 density is likely to at least in part underlie the mechanical hypersensitivity and pain of sharp, shooting quality that occurs in association with inflammation. Indeed, Nav 1.7 is a likely target for the activity-dependent analgesic effects of ranolazine seen following CFA injection [21,49,50].
Discussion Ranolazine reduced the mechanical allodynia produced by the injection of the anthracycline, anti-neoplastic antibiotic doxorubicin into the sciatic nerve. This occurred at doses that did not produce behavioral side effects such as ataxia. This observation is consistent with our previous studies that demonstrated that ranolazine decreased mechanical allodynia in the CFA and SNI preclinical models of inflammatory and neuropathic pain, respectively. Across these animal models, we note that ranolazine provides modality-specific analgesia for mechanical allodynia. These data are evidence that ranolazine is likely to be effective in reducing mechanical
Figure 4 Intraperitoneal ranolazine (30 mg/kg) does not change rotarod latency, a measure of ataxia (N = 3/group). Ranolazine only produced ataxia at the highest dose of 100 mg/kg. A significance level of P < 0.05 is indicated by the asterisk. i.p. = intraperitoneal.
Similarly, the Nav 1.7 and 1.8 isoforms appear to contribute to the hypersensitivity that develops following peripheral nerve injury. The rapid re-priming of Nav 1.8 channels in uninjured dorsal root ganglion neurons, the increased amplitude of the Nav 1.7 and 1.8 currents [51], and the recovery from inactivation of the Nav 1.7 channel [52,53], coupled with after-discharges mediated through transient receptor potential V1–positive fibers, may also contribute to the mechanical allodynia associated with SNI through the augmentation of activity of the Nav 1.8 isoforms [52,54–57] and the accelerated recovery from inactivation of the tetrodotoxin-sensitive currents, isoforms Nav 1.3 and 1.7. The Nav 1.3 isoform is, however, less likely to have a significant impact on SNIinduced hypersensitivity because Nav 1.3 decreases after injury, and anti-sense for Nav 1.3 production has been shown to have no effect on hypersensitivity [58]. Thus, Nav 1.7 and 1.8 are likely targets for the analgesic effects of ranolazine in the neuropathic pain seen following nerve injury [52,59,60]. By contrast, the way ranolazine confers its analgesic effect following demyelination injury is not as readily apparent because demyelination is more closely associated with changes in the Nav 1.3 and 1.6 sodium channel isoforms. Doxorubicin is a topoisomerase inhibitor that is a used as chemotherapeutic agent for the treatment of ovarian cancer, AIDSrelated Kaposi’s sarcoma, and multiple myeloma [61]. When doxorubicin is administered in small amounts, 1775
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
compared with pre-doxorubicin baseline, and a measure of effect size (partial η2) determined that approximately 28% of the variability in withdrawal thresholds was explained by the doxorubicin injection.
Gould et al. 1.8. This study provides evidence that ranolazine selectively attenuates painful mechanical hypersensitivity associated with the i.n. injection of doxorubicin. Further investigation of ranolazine as an option for managing pain associated with any mechanism of injury may be warranted. Acknowledgments HJG and DP were supported by the Department of Pharmacology and Experimental Therapeutics and Department of Neurology at the LSU Health Sciences Center in New Orleans and a grant from Gilead Sciences Inc. and the Neuropathy Association. BKT was supported by National Institutes of Health (R01NS045954). References 1 Hodgkin AL, Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 1952;117(4): 500–44. 2 Cummins TR, Sheets PL, Waxman SG. The roles of sodium channels in nociception: Implications for mechanisms of pain. Pain 2007;131(3):243– 57. 3 Devor M, Seltzer Z. The pathophysiology of damaged peripheral nerves. In: Wall P, Melzack R, eds. Textbook of Pain, 4th edition. Edinburgh: Churchill Livingstone; 1999:129–64. 4 Devor M, Lomazov P, Matzner O. Sodium channel accumulation in injured axons as a substrate for neuropathic pain. In: Boivie J, Hansson P, Lindblom U, eds. Touch, Temperature, and Pain in Health and Disease: Mechanisms and Assessments. Progress in Pain Research and Management, 3rd edition. Seattle: IASP Press; 1994:207–30. 5 Gould HJ 3rd, England J, Paul D. The modulation of sodium channels during inflammation. In: Krames E, Reig E, eds. The Management of Acute and Chronic Pain: The Use of the “Tools of the Trade.” Proceedings of: “Worldwide Pain Conference”. Bologna, Italy: Monduzzi Editore International Proceedings Division; 2000:27–34. 6 Gould HJ 3rd, Gould TN, Paul D, et al. Development of inflammatory hypersensitivity and augmentation of sodium channels in rat dorsal root ganglia. Brain Res 1999;824(2):296–9.
Conclusion
7 Gould HJ 3rd, Gould TN, England JD, et al. A possible role for nerve growth factor in the augmentation of sodium channels in models of chronic pain. Brain Res 2000;854(1–2):19–29.
Ranolazine attenuates angina by improving cardiac perfusion. The mechanism of action involves a modulatory effect on sodium channel subtypes Nav 1.4, 1.5, 1.7, and
8 Gould HJ 3rd, England JD, Soignier RD, et al. Ibuprofen blocks changes in Na v 1.7 and 1.8 sodium channels associated with complete Freund’s
1776
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
0.19–0.38 μg, by microinjection directly into a nerve bundle [25,62], the toxic effects are largely restricted to Schwann cells producing a subacute, focal, and reversible area of demyelination that spares axons. In the areas of demyelination, the intimate relationship between the Schwann cell and its underlying axon is destroyed, leaving relatively large portions of axonal membrane exposed and susceptible to intra-membrane environmental modification and direct contact between adjacent axons [63,64]. Without myelin, excitable membranes are found in close proximity, allowing for the establishment of ephaptic connections and cross-talk between neurons, which compromises the integrity of a transmitted signal and makes it possible for dispersion and amplification of signals that enter the central nervous system [65]. Additionally, the number of sodium channels increases dramatically and is inserted into the exposed membranes [16,25,62,64,66–70], resulting in the generation of ectopic discharges. The resulting increase in regional excitability is thought to be associated with the perception of tingling, burning, sharp, and shocking pain [71]. In contrast to CFA and SNI where the maintenance or augmentation of the Nav 1.3, 1.7, 1.8, and 1.9 channel isoforms comprises the major post-injury change [49,50,66,72,73], the channel isoforms that populate the demyelinated membrane are the nodal Nav 1.6 subtype and a re-expression of an embryonic form Nav 1.3 [26,74,75], but see Black et al. [76]. Although the additional Nav 1.3 and 1.6 channels would logically be responsible for increased excitability and allodynia following demyelination, these channel isoforms do not provide a target for ranolazine and thus do not explain the analgesic effect of ranolazine observed in our study. However, doxorubicin in doses used in this model produces little, if any, damage to axons [25,26] and thus would have little, if any, effect on unmyelinated axons. The potential for axo-axonal cross-excitation through ephaptic spread between C-fibers [3,4,46,71,77–85], characteristically found to express high levels of the Nav 1.7 and 1.8 channels [46,76,86–88], and the axons of small demyelinated and mechanically sensitive A-β and A-δ axons [46,74,89–91] may well contribute to at least a small portion of the mechanical allodynia and spontaneous excitation that occurs at the site of demyelination. We speculate that the reduction of the ephaptic initiating potential of the C-fiber with ranolazine blockade of Nav 1.7 and 1.8 could explain the analgesia and the magnitude of the effect observed with ranolazine treatment. Furthermore, blockade of Nav 1.7 and 1.8 sodium channels could potentially reduce the efficiency of ephaptically imposed current sinks between A-β and A-δ axons and C-fibers [92–94], thereby improving impulse conduction through the zone of demyelination [95,96] and restoring conduction velocities as observed following the administration of ranolazine.
Analgesic Effects of Ranolazine Treatment adjuvant-induced inflammation 2004;5(5):270–80.
in
rat.
J
Pain
Nav1.7 voltage-gated Na+ channel isoforms by ranolazine. Mol Pharmacol 2008;73(3):940–8.
9 Kral MG, Xiong Z, Study RE. Alteration of Na+ currents in dorsal root ganglion neurons from rats with a painful neuropathy. Pain 1999;81(1–2):15–24.
22 Casey GP, Roberts JS, Paul D, Diamond I, Gould H 3rd. Ranolazine attenuation of CFA-induced mechanical hyperalgesia. Pain Med 2010;11(1):119– 26. 23 Gould HJ 3rd, Garrett C, Donahue RR, et al. Ranolazine attenuates behavioral signs of neuropathic pain. Behav Pharmacol 2009;20(8):755–8.
11 Waxman SG, Dib-Hajj S, Cummins TR, Black JA. Sodium channels and pain. Proc Natl Acad Sci U S A 1999;96(14):7635–9.
24 England JD, Rhee EK, Said G, Sumner AJ. Schwann cell degeneration induced by doxorubicin (adriamycin). Brain 1988;111(Pt 4):901–13.
12 Paul D, Gould HJ 3rd, Dunne-Reilly A Predictive validity of a model of peripheral nerve demyelinationinduced neuropathic pain. 12th World Congress on Pain 2008:PW40.
25 England JD, Gamboni F, Ferguson MA, Levinson SR. Sodium channels accumulate at the tips of injured axons. Muscle Nerve 1994;17(6):593–8.
13 Zimmermann M. Pathobiology of neuropathic pain. Eur J Pharmacol 2001;429(1–3):23–37. 14 Erichsen HK, Hao JX, Xu XJ, Blackburn-Munro G. A comparison of the antinociceptive effects of voltageactivated Na+ channel blockers in two rat models of neuropathic pain. Eur J Pharmacol 2003;458(3): 275–82. 15 Decosterd I, Allchorne A, Woolf CJ. Differential analgesic sensitivity of two distinct neuropathic pain models. Anesth Analg 2004;99(2):457–63. Table of contents. 16 England JD, Happel LT, Kline DG, et al. Sodium channel accumulation in humans with painful neuromas. Neurology 1996;47(1):272–6. 17 Pham DQ, Mehta M. Ranolazine: A novel agent that improves dysfunctional sodium channels. Int J Clin Pract 2007;61(5):864–72. 18 Sossalla S, Wagner S, Rasenack EC, et al. Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts—Role of late sodium current and intracellular ion accumulation. J Mol Cell Cardiol 2008;45(1):32–43. 19 Belardinelli L, Shryock JC, Fraser H. Inhibition of the late sodium current as a potential cardioprotective principle: Effects of the late sodium current inhibitor ranolazine. Heart 2006;92(suppl 4):iv6–14. 20 Rajamani S, Shryock JC, Belardinelli L. Block of tetrodotoxin-sensitive, Na(V)1.7 and tetrodotoxinresistant, Na(V)1.8, Na+ channels by ranolazine. Channels (Austin) 2008;2(6):449–60. 21 Wang GK, Calderon J, Wang SY. State- and usedependent block of muscle Nav1.4 and neuronal
26 Paul D, Dunne-Reilly A, England J, Gould HJ 3rd. Sodium channel modulation in an animal model of peripheral nerve demyelination. J Pain 2009;10:S22. 27 Gould HJ 3rd, England J, Liu Z, Levinson S. Sodium channel augmentation in response to pain induced by chronic inflammation. Am Pain Soc 1997;16:99. 28 Gould HJ 3rd, England JD, Liu ZP, Levinson SR. Rapid sodium channel augmentation in response to inflammation induced by complete Freund’s adjuvant. Brain Res 1998;802(1–2):69–74. 29 Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32(1):77–88. 30 Lewin GR, Ritter AM, Mendell LM. Nerve growth factor-induced hyperalgesia in the neonatal and adult rat. J Neurosci 1993;13(5):2136–48. 31 Lewin GR, Rueff A, Mendell LM. Peripheral and central mechanisms of NGF-induced hyperalgesia. Eur J Neurosci 1994;6(12):1903–12. 32 Soignier RD, Taylor BK, Baiamonte BA, et al. Measurement of CFA-induced hyperalgesia and morphine-induced analgesia in rats: Dorsal vs plantar mechanical stimulation of the hindpaw. Pain Med 2011;12(3):451–8. 33 Bouhassira D, Attal N, Willer JC, Brasseur L. Painful and painless peripheral sensory neuropathies due to HIV infection: A comparison using quantitative sensory evaluation. Pain 1999;80(1–2):265–72. 34 Spray DC, Dermietzel R. X-linked dominant CharcotMarie-Tooth disease and other potential gap-junction diseases of the nervous system. Trends Neurosci 1995;18(6):256–62. 1777
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
10 Waxman SG, Cummins TR, Dib-Hajj SD, Black JA. Voltage-gated sodium channels and the molecular pathogenesis of pain: A review. J Rehabil Res Dev 2000;37(5):517–28.
Gould et al. 35 Zhang JL, Yang JP, Zhang JR, et al. Gabapentin reduces allodynia and hyperalgesia in painful diabetic neuropathy rats by decreasing expression level of Nav1.7 and p-ERK1/2 in DRG neurons. Brain Res 2013;1493:13–8.
48 Luo S, Perry GM, Levinson SR, Henry MA. Pulpitis increases the proportion of atypical nodes of Ranvier in human dental pulp axons without a change in Nav1.6 sodium channel expression. Neuroscience 2010;169(4):1881–7.
36 Marx C, Sweeney M. Mechanism of action of ranolazine. Arch Intern Med 2006;166(12): 1325–6, author reply 1326.
49 Lolignier S, Amsalem M, Maingret F, et al. Nav1.9 channel contributes to mechanical and heat pain hypersensitivity induced by subacute and chronic inflammation. PLoS ONE 2011;6(8):e23083.
37 Nash DT. The case for medical treatment in chronic stable coronary artery disease. Arch Intern Med 2005;165(22):2587–9, discussion 2593–4.
39 Haufe V, Camacho JA, Dumaine R, et al. Expression pattern of neuronal and skeletal muscle voltage-gated Na+ channels in the developing mouse heart. J Physiol 2005;564(Pt 3):683–96. 40 Ju YK, Saint DA, Gage PW. Hypoxia increases persistent sodium current in rat ventricular myocytes. J Physiol 1996;497(Pt 2):337–47. 41 Ju YK, Gage PW, Saint DA. Tetrodotoxin-sensitive inactivation-resistant sodium channels in pacemaker cells influence heart rate. Pflugers Arch 1996;431(6): 868–75. 42 Baetz D, Bernard M, Pinet C, et al. Different pathways for sodium entry in cardiac cells during ischemia and early reperfusion. Mol Cell Biochem 2003;242(1– 2):115–20. 43 Song Y, Shryock JC, Wagner S, Maier LS, Belardinelli L. Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction. J Pharmacol Exp Ther 2006; 318(1):214–22. 44 Song Y, Shryock JC, Belardinelli L. An increase of late sodium current induces delayed after depolarizations and sustained triggered activity in atrial myocytes. Am J Physiol Heart Circ Physiol 2008;294(5):H2031–9. 45 Zaza A, Belardinelli L, Shryock JC. Pathophysiology and pharmacology of the cardiac “late sodium current”. Pharmacol Ther 2008;119(3):326–39. 46 Levinson SR, Luo S, Henry MA. The role of sodium channels in chronic pain. Muscle Nerve 2012;46(2): 155–65. 47 Luo S, Perry GM, Levinson SR, Henry MA. Nav1.7 expression is increased in painful human dental pulp. Mol Pain 2008;4:16. doi:10.1186/1744-8069-416. 1778
51 Laedermann CJ, Cachemaille M, Kirschmann G, et al. Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain. J Clin Invest 2013;123(7):3002–13. 52 Berta T, Poirot O, Pertin M, et al. Transcriptional and functional profiles of voltage-gated Na(+) channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain. Mol Cell Neurosci 2008;37(2):196–208. 53 Suter MR, Kirschmann G, Laedermann CJ, Abriel H, Decosterd I. Rufinamide attenuates mechanical allodynia in a model of neuropathic pain in the mouse and stabilizes voltage-gated sodium channel inactivated state. Anesthesiology 2013;118(1):160–72. 54 Goldin AL. Resurgence of sodium channel research. Annu Rev Physiol 2001;63:871–94. 55 Goldin AL. Mechanisms of sodium channel inactivation. Curr Opin Neurobiol 2003;13(3):284–90. 56 Pertin M, Ji RR, Berta T, et al. Upregulation of the voltage-gated sodium channel beta2 subunit in neuropathic pain models: Characterization of expression in injured and non-injured primary sensory neurons. J Neurosci 2005;25(47):10970–80. 57 Lopez-Santiago LF, Pertin M, Morisod X, et al. Sodium channel beta2 subunits regulate tetrodotoxin-sensitive sodium channels in small dorsal root ganglion neurons and modulate the response to pain. J Neurosci 2006;26(30):7984–94. 58 Lindia JA, Kohler MG, Martin WJ, Abbadie C. Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats. Pain 2005;117(1–2):145–53. 59 Yamamoto S, Ohsawa M, Ono H. Contribution of TRPV1 receptor-expressing fibers to spinal ventral root after-discharges and mechanical hyperalgesia in a spared nerve injury (SNI) rat model. J Pharmacol Sci 2013;121(1):9–16.
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
38 Hale SL, Shryock JC, Belardinelli L, Sweeney M, Kloner RA. Late sodium current inhibition as a new cardioprotective approach. J Mol Cell Cardiol 2008;44(6):954–67.
50 Shields SD, Cheng X, Uceyler N, et al. Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury. J Neurosci 2012;32(32):10819–32.
Analgesic Effects of Ranolazine Treatment 60 Yoon YW, Na HS, Chung JM. Contributions of injured and intact afferents to neuropathic pain in an experimental rat model. Pain 1996;64(1):27–36. 61 Physicians’ Desk Reference, 67th edition. Montvale NJ: PDR Network LLC; 2013. 62 England JD, Gamboni F, Levinson SR, Finger TE. Changed distribution of sodium channels along demyelinated axons. Proc Natl Acad Sci U S A 1990;87(17):6777–80. 63 Bray GM, Rasminsky M, Aguayo AJ. Interactions between axons and their sheath cells. Annu Rev Neurosci 1981;4:127–62.
65 Jefferys JG. Nonsynaptic modulation of neuronal activity in the brain: Electric currents and extracellular ions. Physiol Rev 1995;75(4):689–723. 66 Black JA, Nikolajsen L, Kroner K, Jensen TS, Waxman SG. Multiple sodium channel isoforms and mitogenactivated protein kinases are present in painful human neuromas. Ann Neurol 2008;64(6):644–53. 67 England JD, Gamboni F, Levinson SR. Increased numbers of sodium channels form along demyelinated axons. Brain Res 1991;548(1–2):334–7. 68 Henry MA, Freking AR, Johnson LR, Levinson SR. Increased sodium channel immunofluorescence at myelinated and demyelinated sites following an inflammatory and partial axotomy lesion of the rat infraorbital nerve. Pain 2006;124(1–2):222–33. 69 Henry MA, Luo S, Foley BD, et al. Sodium channel expression and localization at demyelinated sites in painful human dental pulp. J Pain 2009;10(7): 750–8. 70 Henry MA, Luo S, Levinson SR. Unmyelinated nerve fibers in the human dental pulp express markers for myelinated fibers and show sodium channel accumulations. BMC Neurosci 2012;13:29. doi: 10.1186/ 1471-2202-13-29. 71 Attal N, Bouhassira D. Mechanisms of pain in peripheral neuropathy. Acta Neurol Scand Suppl 1999;173:12–24, discussion 48–52. 72 Decosterd I, Ji RR, Abdi S, Tate S, Woolf CJ. The pattern of expression of the voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 does not change in uninjured primary sensory neurons in experimental neuropathic pain models. Pain 2002;96(3): 269–77.
74 Caldwell JH, Schaller KL, Lasher RS, Peles E, Levinson SR. Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses. Proc Natl Acad Sci U S A 2000;97(10):5616–20. 75 Henry MA, Freking AR, Johnson LR, Levinson SR. Sodium channel Nav1.6 accumulates at the site of infraorbital nerve injury. BMC Neurosci 2007;8:56. 76 Black JA, Frezel N, Dib-Hajj SD, Waxman SG. Expression of Nav1.7 in DRG neurons extends from peripheral terminals in the skin to central preterminal branches and terminals in the dorsal horn. Mol Pain 2012;8:82. doi: 10.1186/1744-8069-8-82. 77 Fried K, Govrin-Lippmann R, Devor M. Close apposition among neighbouring axonal endings in a neuroma. J Neurocytol 1993;22(8):663–81. 78 Lisney SJ, Pover CM. Coupling between fibres involved in sensory nerve neuromata in cats. J Neurol Sci 1983;59(2):255–64. 79 Rasminsky M. Pathophysiology of demyelination. Ann N Y Acad Sci 1984;436:68–85. 80 Rasminsky M. Ectopic generation of impulses and cross-talk in spinal nerve roots of “dystrophic” mice. Ann Neurol 1978;3(4):351–7. 81 Rasminsky M. Ephaptic transmission between single nerve fibres in the spinal nerve roots of dystrophic mice. J Physiol 1980;305:151–69. 82 Rasminsky M. Ectopic excitation, ephaptic excitation and autoexcitation in peripheral nerve fibers of mutant mice. In: Culp W, Ochoa J, eds. Abnormal Nerves and Muscles as Impulse Generators. Oxford: Oxford University Press; 1982:344–62. 83 Rasminsky M. Spontaneous activity and cross-talk in pathological nerve fibers. In: Kandel E, ed. Molecular Neurobiology in Neurology and Psychiatry. New York: Raven Press; 1987:39–49. 84 Seltzer Z, Devor M. Ephaptic transmission in chronically damaged peripheral nerves. Neurology 1979;29(7):1061–4. 85 Sun QQ. A novel role of dendritic gap junction and mechanisms underlying its interaction with thalamocortical conductance in fast spiking inhibitory neurons. BMC Neurosci 2009;10:131. doi: 10.1186/14712202-10-131. 1779
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
64 Dugandzija-Novakovic S, Koszowski AG, Levinson SR, Shrager P. Clustering of Na+ channels and node of Ranvier formation in remyelinating axons. J Neurosci 1995;15(1 Pt 2):492–503.
73 Kretschmer T, Happel LT, England JD, et al. Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry. Acta Neurochir (Wien) 2002;144(8):803–10, discussion 810.
Gould et al. 86 Amaya F, Decosterd I, Samad TA, et al. Diversity of expression of the sensory neuron-specific TTXresistant voltage-gated sodium ion channels SNS and SNS2. Mol Cell Neurosci 2000;15(4):331–42.
91 Bonica J. Anatomic and physiologic basis of nociception and pain. In: Bonica J, ed. The Management of Pain, 2nd edition. Philadelphia: Lea & Febiger; 1990:28–94.
87 Djouhri L, Newton R, Levinson SR, et al. Sensory and electrophysiological properties of guinea-pig sensory neurones expressing Nav 1.7 (PN1) Na+ channel alpha subunit protein. J Physiol 2003;546(Pt 2):565– 76.
92 Harris AL, Spray DC, Bennett MV. Kinetic properties of a voltage-dependent junctional conductance. J Gen Physiol 1981;77(1):95–117.
88 Toledo-Aral JJ, Moss BL, He ZJ, et al. Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons. Proc Natl Acad Sci U S A 1997;94(4):1527–32.
93 Loewenstein WR. Junctional intercellular communication: The cell-to-cell membrane channel. Physiol Rev 1981;61(4):829–913. 94 Spray DC, Harris AL, Bennett MV. Equilibrium properties of a voltage-dependent junctional conductance. J Gen Physiol 1981;77(1):77–93. 95 Bostock H, Sears TA. Continuous conduction in demyelinated mammalian nerve fibers. Nature 1976; 263(5580):786–7.
90 Andrew D, Greenspan JD. Peripheral coding of tonic mechanical cutaneous pain: Comparison of nociceptor activity in rat and human psychophysics. J Neurophysiol 1999;82(5):2641–8.
96 Bostock H, Sears TA. The internodal axon membrane: Electrical excitability and continuous conduction in segmental demyelination. J Physiol 1978;280: 273–301.
1780
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
89 Andrew D, Greenspan JD. Mechanical and heat sensitization of cutaneous nociceptors after peripheral inflammation in the rat. J Neurophysiol 1999; 82(5):2649–56.
Pain Medicine 2014; 15: 1771–1780 Wiley Periodicals, Inc.
NEUROPATHIC PAIN SECTION Original Research Article Ranolazine Attenuates Mechanical Allodynia Associated with Demyelination Injury
Departments of *Physical Medicine and Rehabilitation, † Neurology, ††Pharmacology and Experimental Therapeutics, ‡Pain Mastery Center of Louisiana, Louisiana State University Health Sciences Center, New Orleans, Louisiana; §Department of Psychology, Nicholls State University, Thibodaux, Louisiana; ¶ Gilead Sciences Inc., Palo Alto, California; **Department of Physiology, University of Kentucky Medical Center, Lexington, Kentucky, USA Reprint requests to: Harry J. Gould, III, MD, PhD, Department of Neurology, LSU Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA. Tel: 504-568-4080; Fax: 504-568-7130; E-mail: [email protected]. Disclosure: ID is a senior advisor at Gilead Sciences Inc., producer of ranolazine.
of this drug in a newly validated model of demyelination injury that responds uniquely to a number of treatment options. Methods. After determination of baseline nerve conduction velocities (NCVs) and withdrawal responses from heat and mechanical stimulation in male Sprague-Dawley rats (300–350 g), 1 μg/30 μL of doxorubicin was injected into one sciatic nerve. The contralateral nerve provided a sham-injected control. Two weeks after doxorubicin injection, NCV and sensitivity to heat and mechanical stimulation were reassessed before and after treatment with ranolazine (10, 30, 50 mg/kg) administered intraperitoneally using an experimenter-blinded, randomized design. Results. Doxorubicin injection produced a significant hyperalgesic effect in response to mechanical but not heat stimulation. Conduction velocities in the injected limbs were reduced when compared with controls. Ranolazine reduced mechanical allodynia with peak efficacy at 30 mg/kg. Fifty milligram/kilogram ranolazine restored NCVs by approximately 50%, but had no effect in the uninjected limb.
Abstract
Conclusions. Ranolazine exerts broad-spectrum actions to reduce mechanical allodynia that is associated with peripheral demyelination injury.
Objective. The aim of this study was to determine whether ranolazine, a new medication that targets sodium channels to improve cardiac ischemia and angina, could be an effective analgesic agent for pain associated with demyelination injury.
Key Words. Ranolazine; Allodynia
Background. Many agents have been used to treat neuropathic pain but not all neuropathic conditions respond similarly to treatment. We have demonstrated that ranolazine, an agent that blocks voltagegated sodium channels Nav 1.4, 1.5, 1.7, and 1.8, is effective in attenuating mechanical hyperalgesia in both complete Freund’s adjuvant and spared nerve injury preclinical models of inflammatory and neuropathic pain, respectively. Here we test the efficacy
Doxorubicin;
Pain;
Introduction Sodium channels are responsible for the generation and propagation of electrochemical excitability and thus have a significant influence on the function of excitable cells [1]. Injury changes the expression of sodium channels in peripheral nerves. These changes are well correlated with the perception and reporting of pain [2–11], but the patterns of hypersensitivity associated with particular disease 1771
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
Harry J. Gould, III, MD, PhD,*,†,‡ R. Denis Soignier, PhD,‡,§ Sung R. Cho, MD,*,‡ Christian Hernandez, MD,†,‡ Ivan Diamond, MD, PhD,¶ Bradley K. Taylor, PhD,** and Dennis Paul, PhD†,‡,††
Gould et al. states and their response to treatment vary significantly both in preclinical and clinical settings [12–15]. These differences confound our attempts to provide consistent care for patients with pain. Because of the consistent association between hyperalgesia and alterations in sodium channel distribution and density [16], sodium channel blockers are frequently used to produce analgesia for conditions arising from a number of disease states and mechanisms of injury.
Methods Animals All experiments were conducted in accordance with protocols that were approved and monitored by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN, USA) weighing between 300 and 350 g were housed, one animal to a cage, and maintained at 25°C and 60% humidity, on a 12-hour light/dark cycle and allowed access to food and water ad libitum. For 1 week, rats were allowed to acclimate to their surroundings and for 1 hour/day to the testing apparatus. 1772
To determine baseline thresholds to heat stimulation, rats from groups of nine were placed in Plexiglas chambers on a glass plate and were allowed free range of activity within the chamber. The glabrous surface of each hind paw was stimulated sequentially through the glass plate using a halogen light source [27–29]. The latency of paw withdrawal from the onset of stimulation was measured using an IITC analgesiometer (IITC Life Science, Inc., Woodland Hills, CA, USA). The stimulus was automatically discontinued after 10.7 seconds to avoid tissue damage. Each hind paw was stimulated four times during each testing session. The interval between stimulation of paws in a given rat was approximately 2 minutes and no less than 1 minute between trials.
Behavioral Assessment of Mechanical Sensitivity Approximately 30 minutes after heat testing, thresholds to withdrawal from mechanical stimulation were assessed using an IITC Model 2290 electro-von Frey (EVF) anesthesiometer (IITC Life Science, Inc.) [30–32]. For this, the rats were loosely restrained in a cotton towel and allowed to accommodate to the restriction. The tip of the stimulating wand was then applied perpendicular to the skin at four sites on the dorsal surface of each hind paw. The force applied to the paw at the time of paw withdrawal was recorded. The average force applied to the four sites was entered as the subject’s response threshold for the interval and used in all further calculations. A ceiling of 250 g of force was imposed to prevent tissue injury from EVF testing.
CMAPs Finally, after establishing baseline withdrawal thresholds from heat and mechanical stimulation, baseline CMAPs were ascertained in both hind extremities using a NeuroMax1000 unit (Excel Tech, Ltd., Oakville, ON, Canada) to calculate sciatic nerve conduction velocity (NCV). Twenty-seven-gauge, 1.5-cm subdermal needle recording electrodes (CareFusion Corporation, San Diego, CA, USA) were inserted between third and fourth digits of the hind paw. Stimulation of variable duration and voltage to elicit optimal responses was applied distally at the calcaneal tendon and proximally just inferior to the hip joint. The ground electrode was placed between the stimulating and recording sites, and the distance between the stimulating and recording electrodes was measured. The CMAP amplitude and duration and the latency between the stimulus artifact and the initial deflection from the baseline trace were measured and the NCV was calculated. Conduction block was inferred by recording a greater than 10 m/second or 30% decrease in the conduction velocity of the sciatic nerve from baseline values. Due to significant variation in CMAP amplitudes, only NCV criteria were used to confirm demyelination during baseline recording.
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
One such agent is ranolazine (Ranexa®, Gilead Science Inc., Palo Alto, CA, USA), which has been approved by the U.S. Food and Drug Administration (FDA) for treating cardiac ischemia and anginal chest pain [17,18]. The antianginal effects of ranolazine are produced by blocking the late sodium current associated with the voltage-gated sodium channel Nav 1.5. In addition, ranolazine use dependently inhibits Nav 1.7 and 1.8, sodium channels that are linked to the regulation of inflammatory and/or neuropathic pain [19–21]. Because of its affinity for specific sodium channels, we previously tested ranolazine in both the complete Freund’s adjuvant (CFA) [22] and spared nerve injury (SNI) [23] preclinical models of inflammatory and neuropathic pain, respectively. We demonstrated that ranolazine attenuated mechanical and cold hypersensitivity [23], but did not reduce hypersensitivity associated with heat [22]. Because of the consistent analgesic effect on mechanical hyperalgesia present in both inflammatory and neuropathic conditions, we hypothesized that ranolazine would attenuate the mechanical allodynia produced by intraneural (i.n.) injection of doxorubicin, a DNA-intercalating agent that causes delayed subacute, focal, and reversible demyelination by killing Schwann cells and sparing axons [24], analogous to that seen in Guillain–Barré syndrome and chronic inflammatory demyelinating polyneuropathy. This newly validated model of demyelination injury alters the normal distribution and density of sodium channels in the area of the injection site, thereby compromising salutatory conduction of action potentials and resulting in conduction slowing, a common feature of demyelination injury, a reduction in the compound motor action potential (CMAP) [24,25], and a disruption of sensory processing giving rise to an increase in sensitivity to mechanical but not heat stimulation [12,26].
Behavioral Assessment of Heat Hypersensitivity
Analgesic Effects of Ranolazine Treatment Model of Demyelination Injury
Results
Once all the baseline determinations were made, small incisions were made bilaterally at the level of the sciatic notch to expose the sciatic nerves. One sciatic nerve received a 1 μg/30 μL i.n. injection of doxorubicin (Adriamycin®, Sigma-Aldrich, St. Louis, MO, USA). The contralateral nerve was injected with an equal volume of normal saline and served as a sham control. The electrodiagnostician was blinded to which nerve received doxorubicin.
A single injection of doxorubicin into the main trunk of a sciatic nerve produces electrodiagnostic evidence of conduction slowing within 2 weeks of the injection (Figures 1 and 2). The average conduction velocity in the doxorubicin-injected limb was significantly reduced when compared with the contralateral saline-injected controls, 27.79 m/second vs 39.2 m/second, respectively; ANOVA revealed significant interaction: F(2,24) = 3.99, P > 0.05.
Experimental Design
Behavioral Test of Ataxia To determine whether ranolazine nonspecifically reduced central nervous system function such as motor control, we tested its effects in the rotarod test. As previously described [22,23], the ability of animals to remain on an accelerating rotarod was assessed before and after i.p. administration of vehicle or ranolazine (up to 100 mg/kg). Rats were placed on the rotarod, set at an initial rotating speed of 4 rpm. Immediately thereafter, the speed of the rod increased at a rate of 0.5 rpm every 5 seconds to a maximum of 40 rpm. Each rat was trained in this procedure three to nine times until latency to fall was approximately 180 seconds. Triplicate measurements were taken before and then 30, 60, 90, 120, 150, and 180 minutes after ranolazine injection. Statistics The behavioral data were analyzed using GraphPad Prism for Windows® (Version 5.1, GraphPad Software, Inc., San Diego, CA, USA). Sensory data were analyzed using separate one-way analysis of variance (ANOVA) and conduction velocity data were analyzed using a 2 × 3, mixed design ANOVA to determine statistical significance between experimental conditions. The doxorubicin injection resulted in significant reduction in withdrawal threshold to mechanical stimuli (P < 0.01), but not in withdrawal latency from heat stimuli.
Figure 1 The paired traces depict compound motor action potentials (CMAPs) recorded from rats before (A) and 14 days after (B) the intraneural injection of doxorubicin. The conduction velocity (CV) of the sciatic nerve measured 33.9+/− 2.29 m/second at baseline and then 27.79+/− 2.25 m/second following doxorubicin injection. The greater than 30% decrease in CV is evidence of conduction slowing associated with acute demyelination injury. The paired traces recorded from post-doxorubicin injected sciatic nerves after intraperitoneal (i.p.) administration of 30 mg/kg dosing of ranolazine (C) reveal a CV of 39.4+/− 0.69 m/second, essentially returning CV to baseline levels. NCV = nerve conduction velocity. 1773
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
One to 2 weeks after doxorubicin injection, CMAP determinations were repeated and NCVs were calculated. A greater than 30% reduction in NCVs in the doxorubicin injected limb, 27+/− 2 m/second, when compared with that observed in the contralateral control, 40+/− 3 m/second, provided evidence of successful demyelination. Upon substantiation of demyelination, post-doxorubicin sensitivity to heat and mechanical stimulation was then reassessed on two successive days. On the following day, groups of rats received ranolazine dissolved in 0.9% isotonic saline by intraperitoneal (i.p.) injection (0, 10, 30, and 50 mg/kg) or equal volumes of vehicle in a randomized, experimenter-blinded design. Withdrawal thresholds to heat and mechanical stimulation were reassessed 30 minutes after dosing. Post-ranolazine CMAP determinations were also made after behavioral testing was completed.
Gould et al.
Planned comparison of the effects of doxorubicin injection for each time point using the Bonferroni adjustment was carried out. The analysis revealed that the difference between injured and uninjured paws (Figure 2, gray vs black bars) was only significant at the post-injury time point (P < 0.01). No other significant differences were observed. Neither ranolazine nor doxorubicin injection of the contralateral sciatic nerve significantly changed conduction velocity in the control limb. The amplitudes of the CMAPs were fairly consistent but variable enough in both limbs to preclude determination of the presence or extent of axonal injury that resulted from the doxorubicin injection. Variations in conduction velocity, likely due to variability in estimating the position of the electrode tips when measuring the distance between the stimulating and recording electrodes, were not statistically significant. Consistent with the validation studies of this model [12], the i.n. injection of doxorubicin increased sensitivity to mechanical (F [5,70] = 5.56, P < 0.05), but not heat (F[5,66] = 4.67, P = 0.51) stimulation of the hind paw ipsilateral to the injection. Intraneural doxorubicin did not change the response to heat stimulation applied to the ipsilateral or contralateral paw (Figure 3A). By contrast, Figure 3 illustrates that intraneural (i.n.) doxorubicin increased mechanical sensitivity by approximately 30% as 1774
Figure 3 Left side of graphs depicts the sensitivity to heat (A) and mechanical (B) stimulation before (diagonally lined bars) and 14 days after (crosshatched bars) i.n. doxorubicin (dox) injection. The doxorubicin injection resulted in significant reduction in withdrawal threshold to mechanical stimuli (**P < 0.01), but not in withdrawal latency from heat stimuli. Right side of the graphs indicates the dose– response relationship for 0, 10, 30, and 50 mg/kg intraperitoneal (i.p.) doses of ranolazine. At both 30 and 50 mg/kg, withdrawal thresholds to mechanical stimuli were significantly higher vs postinjection baseline (* = P < 0.05). The maximum reduction of mechanical sensitivity observed following ranolazine administration was achieved at the 30 mg/kg dose but did not return to baseline levels. No additional benefit was observed at higher doses. No significant effects of ranolazine were observed on withdrawal latencies from heat stimuli (A) or on contralateral paw sensitivity (data not shown). i.n. = intraneural.
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
Figure 2 The graphs compare the conduction velocities (CVs) of the injected (gray bars) vs contralateral uninjected control (black bars) sciatic nerves recorded from rats prior to doxorubicin injection (baseline) and 14 days after injection both before (post-dox) and after ranolazine treatment (ranolazine) (asterisk denotes significance between black and gray bars at a given time point, P < 0.01). The CV that decreased by approximately 30% of baseline following doxorubicin injection was restored to baseline after 30 mg/kg dosing of ranolazine. Ranolazine had no significant effect on CV in the control nerves.
Analgesic Effects of Ranolazine Treatment allodynia in painful conditions regardless of the mechanism of injury [12,33–35]. Further investigation of ranolazine as an analgesic adjuvant treatment option for this purpose is warranted.
The i.p. administration of ranolazine (0, 10, 30, and 50 mg/kg), but not vehicle, dose-dependently reduced mechanical allodynia produced by doxorubicin injection (ED50 = 8.14 mg/kg ± 3.1; P < 0.001). Ranolazine reduced allodynia by approximately 60% at the maximally effective dose of 30 mg/kg, which does not impair motor control (Figure 4 and see Casey et al. and Gould et al. [22,23]). At higher doses of 50 mg/kg, ranolazine restored NCVs by approximately 50%. Post hoc statistical comparisons made using Dunnett’s test found significant differences between the doxorubicin-injected paws prior to ranolazine (Figure 3, cross-hatched bars) and the shamtreated baseline (P < 0.01) as well as both 30 mg and 50 mg doses of ranolazine (P < 0.05).
Ranolazine is an FDA-approved medication that targets late sodium currents, thereby improving cardiac perfusion and reducing angina [17,18,36,37]. Ranolazine has a modulatory effect on sodium channel subtypes Nav 1.4, 1.5, 1.7, and 1.8 [38–41] and reduces myocardial injury by decreasing the entry of sodium into cardiac myocytes and by reducing the subsequent influx of calcium via the Ca2+/ Na+ exchanger that occurs during ischemia and early reperfusion [19,20,38,42–45]. Because ranolazine targets the Nav 1.7 and 1.8 isoforms of voltage-gated sodium channels that play a significant role in CFA- and SNIinduced mechanical allodynia, the mechanism for the observed analgesic effect seems intuitive [8,22,23,28]. Inflammation is associated with increased expression of Nav 1.3, 1.7, 1.8, and 1.9 isoforms [46]. For example, subcutaneous injection of CFA produces a rapid and dramatic upregulation of the Nav 1.7 isoform of the voltagegated sodium channel in both the large and small neuronal populations in the dorsal root ganglia that innervate the injection site [6,8,28], as well as in the peripheral nerve endings of primary afferents [47,48]. The increase in Nav 1.7 density is likely to at least in part underlie the mechanical hypersensitivity and pain of sharp, shooting quality that occurs in association with inflammation. Indeed, Nav 1.7 is a likely target for the activity-dependent analgesic effects of ranolazine seen following CFA injection [21,49,50].
Discussion Ranolazine reduced the mechanical allodynia produced by the injection of the anthracycline, anti-neoplastic antibiotic doxorubicin into the sciatic nerve. This occurred at doses that did not produce behavioral side effects such as ataxia. This observation is consistent with our previous studies that demonstrated that ranolazine decreased mechanical allodynia in the CFA and SNI preclinical models of inflammatory and neuropathic pain, respectively. Across these animal models, we note that ranolazine provides modality-specific analgesia for mechanical allodynia. These data are evidence that ranolazine is likely to be effective in reducing mechanical
Figure 4 Intraperitoneal ranolazine (30 mg/kg) does not change rotarod latency, a measure of ataxia (N = 3/group). Ranolazine only produced ataxia at the highest dose of 100 mg/kg. A significance level of P < 0.05 is indicated by the asterisk. i.p. = intraperitoneal.
Similarly, the Nav 1.7 and 1.8 isoforms appear to contribute to the hypersensitivity that develops following peripheral nerve injury. The rapid re-priming of Nav 1.8 channels in uninjured dorsal root ganglion neurons, the increased amplitude of the Nav 1.7 and 1.8 currents [51], and the recovery from inactivation of the Nav 1.7 channel [52,53], coupled with after-discharges mediated through transient receptor potential V1–positive fibers, may also contribute to the mechanical allodynia associated with SNI through the augmentation of activity of the Nav 1.8 isoforms [52,54–57] and the accelerated recovery from inactivation of the tetrodotoxin-sensitive currents, isoforms Nav 1.3 and 1.7. The Nav 1.3 isoform is, however, less likely to have a significant impact on SNIinduced hypersensitivity because Nav 1.3 decreases after injury, and anti-sense for Nav 1.3 production has been shown to have no effect on hypersensitivity [58]. Thus, Nav 1.7 and 1.8 are likely targets for the analgesic effects of ranolazine in the neuropathic pain seen following nerve injury [52,59,60]. By contrast, the way ranolazine confers its analgesic effect following demyelination injury is not as readily apparent because demyelination is more closely associated with changes in the Nav 1.3 and 1.6 sodium channel isoforms. Doxorubicin is a topoisomerase inhibitor that is a used as chemotherapeutic agent for the treatment of ovarian cancer, AIDSrelated Kaposi’s sarcoma, and multiple myeloma [61]. When doxorubicin is administered in small amounts, 1775
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
compared with pre-doxorubicin baseline, and a measure of effect size (partial η2) determined that approximately 28% of the variability in withdrawal thresholds was explained by the doxorubicin injection.
Gould et al. 1.8. This study provides evidence that ranolazine selectively attenuates painful mechanical hypersensitivity associated with the i.n. injection of doxorubicin. Further investigation of ranolazine as an option for managing pain associated with any mechanism of injury may be warranted. Acknowledgments HJG and DP were supported by the Department of Pharmacology and Experimental Therapeutics and Department of Neurology at the LSU Health Sciences Center in New Orleans and a grant from Gilead Sciences Inc. and the Neuropathy Association. BKT was supported by National Institutes of Health (R01NS045954). References 1 Hodgkin AL, Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 1952;117(4): 500–44. 2 Cummins TR, Sheets PL, Waxman SG. The roles of sodium channels in nociception: Implications for mechanisms of pain. Pain 2007;131(3):243– 57. 3 Devor M, Seltzer Z. The pathophysiology of damaged peripheral nerves. In: Wall P, Melzack R, eds. Textbook of Pain, 4th edition. Edinburgh: Churchill Livingstone; 1999:129–64. 4 Devor M, Lomazov P, Matzner O. Sodium channel accumulation in injured axons as a substrate for neuropathic pain. In: Boivie J, Hansson P, Lindblom U, eds. Touch, Temperature, and Pain in Health and Disease: Mechanisms and Assessments. Progress in Pain Research and Management, 3rd edition. Seattle: IASP Press; 1994:207–30. 5 Gould HJ 3rd, England J, Paul D. The modulation of sodium channels during inflammation. In: Krames E, Reig E, eds. The Management of Acute and Chronic Pain: The Use of the “Tools of the Trade.” Proceedings of: “Worldwide Pain Conference”. Bologna, Italy: Monduzzi Editore International Proceedings Division; 2000:27–34. 6 Gould HJ 3rd, Gould TN, Paul D, et al. Development of inflammatory hypersensitivity and augmentation of sodium channels in rat dorsal root ganglia. Brain Res 1999;824(2):296–9.
Conclusion
7 Gould HJ 3rd, Gould TN, England JD, et al. A possible role for nerve growth factor in the augmentation of sodium channels in models of chronic pain. Brain Res 2000;854(1–2):19–29.
Ranolazine attenuates angina by improving cardiac perfusion. The mechanism of action involves a modulatory effect on sodium channel subtypes Nav 1.4, 1.5, 1.7, and
8 Gould HJ 3rd, England JD, Soignier RD, et al. Ibuprofen blocks changes in Na v 1.7 and 1.8 sodium channels associated with complete Freund’s
1776
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
0.19–0.38 μg, by microinjection directly into a nerve bundle [25,62], the toxic effects are largely restricted to Schwann cells producing a subacute, focal, and reversible area of demyelination that spares axons. In the areas of demyelination, the intimate relationship between the Schwann cell and its underlying axon is destroyed, leaving relatively large portions of axonal membrane exposed and susceptible to intra-membrane environmental modification and direct contact between adjacent axons [63,64]. Without myelin, excitable membranes are found in close proximity, allowing for the establishment of ephaptic connections and cross-talk between neurons, which compromises the integrity of a transmitted signal and makes it possible for dispersion and amplification of signals that enter the central nervous system [65]. Additionally, the number of sodium channels increases dramatically and is inserted into the exposed membranes [16,25,62,64,66–70], resulting in the generation of ectopic discharges. The resulting increase in regional excitability is thought to be associated with the perception of tingling, burning, sharp, and shocking pain [71]. In contrast to CFA and SNI where the maintenance or augmentation of the Nav 1.3, 1.7, 1.8, and 1.9 channel isoforms comprises the major post-injury change [49,50,66,72,73], the channel isoforms that populate the demyelinated membrane are the nodal Nav 1.6 subtype and a re-expression of an embryonic form Nav 1.3 [26,74,75], but see Black et al. [76]. Although the additional Nav 1.3 and 1.6 channels would logically be responsible for increased excitability and allodynia following demyelination, these channel isoforms do not provide a target for ranolazine and thus do not explain the analgesic effect of ranolazine observed in our study. However, doxorubicin in doses used in this model produces little, if any, damage to axons [25,26] and thus would have little, if any, effect on unmyelinated axons. The potential for axo-axonal cross-excitation through ephaptic spread between C-fibers [3,4,46,71,77–85], characteristically found to express high levels of the Nav 1.7 and 1.8 channels [46,76,86–88], and the axons of small demyelinated and mechanically sensitive A-β and A-δ axons [46,74,89–91] may well contribute to at least a small portion of the mechanical allodynia and spontaneous excitation that occurs at the site of demyelination. We speculate that the reduction of the ephaptic initiating potential of the C-fiber with ranolazine blockade of Nav 1.7 and 1.8 could explain the analgesia and the magnitude of the effect observed with ranolazine treatment. Furthermore, blockade of Nav 1.7 and 1.8 sodium channels could potentially reduce the efficiency of ephaptically imposed current sinks between A-β and A-δ axons and C-fibers [92–94], thereby improving impulse conduction through the zone of demyelination [95,96] and restoring conduction velocities as observed following the administration of ranolazine.
Analgesic Effects of Ranolazine Treatment adjuvant-induced inflammation 2004;5(5):270–80.
in
rat.
J
Pain
Nav1.7 voltage-gated Na+ channel isoforms by ranolazine. Mol Pharmacol 2008;73(3):940–8.
9 Kral MG, Xiong Z, Study RE. Alteration of Na+ currents in dorsal root ganglion neurons from rats with a painful neuropathy. Pain 1999;81(1–2):15–24.
22 Casey GP, Roberts JS, Paul D, Diamond I, Gould H 3rd. Ranolazine attenuation of CFA-induced mechanical hyperalgesia. Pain Med 2010;11(1):119– 26. 23 Gould HJ 3rd, Garrett C, Donahue RR, et al. Ranolazine attenuates behavioral signs of neuropathic pain. Behav Pharmacol 2009;20(8):755–8.
11 Waxman SG, Dib-Hajj S, Cummins TR, Black JA. Sodium channels and pain. Proc Natl Acad Sci U S A 1999;96(14):7635–9.
24 England JD, Rhee EK, Said G, Sumner AJ. Schwann cell degeneration induced by doxorubicin (adriamycin). Brain 1988;111(Pt 4):901–13.
12 Paul D, Gould HJ 3rd, Dunne-Reilly A Predictive validity of a model of peripheral nerve demyelinationinduced neuropathic pain. 12th World Congress on Pain 2008:PW40.
25 England JD, Gamboni F, Ferguson MA, Levinson SR. Sodium channels accumulate at the tips of injured axons. Muscle Nerve 1994;17(6):593–8.
13 Zimmermann M. Pathobiology of neuropathic pain. Eur J Pharmacol 2001;429(1–3):23–37. 14 Erichsen HK, Hao JX, Xu XJ, Blackburn-Munro G. A comparison of the antinociceptive effects of voltageactivated Na+ channel blockers in two rat models of neuropathic pain. Eur J Pharmacol 2003;458(3): 275–82. 15 Decosterd I, Allchorne A, Woolf CJ. Differential analgesic sensitivity of two distinct neuropathic pain models. Anesth Analg 2004;99(2):457–63. Table of contents. 16 England JD, Happel LT, Kline DG, et al. Sodium channel accumulation in humans with painful neuromas. Neurology 1996;47(1):272–6. 17 Pham DQ, Mehta M. Ranolazine: A novel agent that improves dysfunctional sodium channels. Int J Clin Pract 2007;61(5):864–72. 18 Sossalla S, Wagner S, Rasenack EC, et al. Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts—Role of late sodium current and intracellular ion accumulation. J Mol Cell Cardiol 2008;45(1):32–43. 19 Belardinelli L, Shryock JC, Fraser H. Inhibition of the late sodium current as a potential cardioprotective principle: Effects of the late sodium current inhibitor ranolazine. Heart 2006;92(suppl 4):iv6–14. 20 Rajamani S, Shryock JC, Belardinelli L. Block of tetrodotoxin-sensitive, Na(V)1.7 and tetrodotoxinresistant, Na(V)1.8, Na+ channels by ranolazine. Channels (Austin) 2008;2(6):449–60. 21 Wang GK, Calderon J, Wang SY. State- and usedependent block of muscle Nav1.4 and neuronal
26 Paul D, Dunne-Reilly A, England J, Gould HJ 3rd. Sodium channel modulation in an animal model of peripheral nerve demyelination. J Pain 2009;10:S22. 27 Gould HJ 3rd, England J, Liu Z, Levinson S. Sodium channel augmentation in response to pain induced by chronic inflammation. Am Pain Soc 1997;16:99. 28 Gould HJ 3rd, England JD, Liu ZP, Levinson SR. Rapid sodium channel augmentation in response to inflammation induced by complete Freund’s adjuvant. Brain Res 1998;802(1–2):69–74. 29 Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988;32(1):77–88. 30 Lewin GR, Ritter AM, Mendell LM. Nerve growth factor-induced hyperalgesia in the neonatal and adult rat. J Neurosci 1993;13(5):2136–48. 31 Lewin GR, Rueff A, Mendell LM. Peripheral and central mechanisms of NGF-induced hyperalgesia. Eur J Neurosci 1994;6(12):1903–12. 32 Soignier RD, Taylor BK, Baiamonte BA, et al. Measurement of CFA-induced hyperalgesia and morphine-induced analgesia in rats: Dorsal vs plantar mechanical stimulation of the hindpaw. Pain Med 2011;12(3):451–8. 33 Bouhassira D, Attal N, Willer JC, Brasseur L. Painful and painless peripheral sensory neuropathies due to HIV infection: A comparison using quantitative sensory evaluation. Pain 1999;80(1–2):265–72. 34 Spray DC, Dermietzel R. X-linked dominant CharcotMarie-Tooth disease and other potential gap-junction diseases of the nervous system. Trends Neurosci 1995;18(6):256–62. 1777
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
10 Waxman SG, Cummins TR, Dib-Hajj SD, Black JA. Voltage-gated sodium channels and the molecular pathogenesis of pain: A review. J Rehabil Res Dev 2000;37(5):517–28.
Gould et al. 35 Zhang JL, Yang JP, Zhang JR, et al. Gabapentin reduces allodynia and hyperalgesia in painful diabetic neuropathy rats by decreasing expression level of Nav1.7 and p-ERK1/2 in DRG neurons. Brain Res 2013;1493:13–8.
48 Luo S, Perry GM, Levinson SR, Henry MA. Pulpitis increases the proportion of atypical nodes of Ranvier in human dental pulp axons without a change in Nav1.6 sodium channel expression. Neuroscience 2010;169(4):1881–7.
36 Marx C, Sweeney M. Mechanism of action of ranolazine. Arch Intern Med 2006;166(12): 1325–6, author reply 1326.
49 Lolignier S, Amsalem M, Maingret F, et al. Nav1.9 channel contributes to mechanical and heat pain hypersensitivity induced by subacute and chronic inflammation. PLoS ONE 2011;6(8):e23083.
37 Nash DT. The case for medical treatment in chronic stable coronary artery disease. Arch Intern Med 2005;165(22):2587–9, discussion 2593–4.
39 Haufe V, Camacho JA, Dumaine R, et al. Expression pattern of neuronal and skeletal muscle voltage-gated Na+ channels in the developing mouse heart. J Physiol 2005;564(Pt 3):683–96. 40 Ju YK, Saint DA, Gage PW. Hypoxia increases persistent sodium current in rat ventricular myocytes. J Physiol 1996;497(Pt 2):337–47. 41 Ju YK, Gage PW, Saint DA. Tetrodotoxin-sensitive inactivation-resistant sodium channels in pacemaker cells influence heart rate. Pflugers Arch 1996;431(6): 868–75. 42 Baetz D, Bernard M, Pinet C, et al. Different pathways for sodium entry in cardiac cells during ischemia and early reperfusion. Mol Cell Biochem 2003;242(1– 2):115–20. 43 Song Y, Shryock JC, Wagner S, Maier LS, Belardinelli L. Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction. J Pharmacol Exp Ther 2006; 318(1):214–22. 44 Song Y, Shryock JC, Belardinelli L. An increase of late sodium current induces delayed after depolarizations and sustained triggered activity in atrial myocytes. Am J Physiol Heart Circ Physiol 2008;294(5):H2031–9. 45 Zaza A, Belardinelli L, Shryock JC. Pathophysiology and pharmacology of the cardiac “late sodium current”. Pharmacol Ther 2008;119(3):326–39. 46 Levinson SR, Luo S, Henry MA. The role of sodium channels in chronic pain. Muscle Nerve 2012;46(2): 155–65. 47 Luo S, Perry GM, Levinson SR, Henry MA. Nav1.7 expression is increased in painful human dental pulp. Mol Pain 2008;4:16. doi:10.1186/1744-8069-416. 1778
51 Laedermann CJ, Cachemaille M, Kirschmann G, et al. Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain. J Clin Invest 2013;123(7):3002–13. 52 Berta T, Poirot O, Pertin M, et al. Transcriptional and functional profiles of voltage-gated Na(+) channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain. Mol Cell Neurosci 2008;37(2):196–208. 53 Suter MR, Kirschmann G, Laedermann CJ, Abriel H, Decosterd I. Rufinamide attenuates mechanical allodynia in a model of neuropathic pain in the mouse and stabilizes voltage-gated sodium channel inactivated state. Anesthesiology 2013;118(1):160–72. 54 Goldin AL. Resurgence of sodium channel research. Annu Rev Physiol 2001;63:871–94. 55 Goldin AL. Mechanisms of sodium channel inactivation. Curr Opin Neurobiol 2003;13(3):284–90. 56 Pertin M, Ji RR, Berta T, et al. Upregulation of the voltage-gated sodium channel beta2 subunit in neuropathic pain models: Characterization of expression in injured and non-injured primary sensory neurons. J Neurosci 2005;25(47):10970–80. 57 Lopez-Santiago LF, Pertin M, Morisod X, et al. Sodium channel beta2 subunits regulate tetrodotoxin-sensitive sodium channels in small dorsal root ganglion neurons and modulate the response to pain. J Neurosci 2006;26(30):7984–94. 58 Lindia JA, Kohler MG, Martin WJ, Abbadie C. Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats. Pain 2005;117(1–2):145–53. 59 Yamamoto S, Ohsawa M, Ono H. Contribution of TRPV1 receptor-expressing fibers to spinal ventral root after-discharges and mechanical hyperalgesia in a spared nerve injury (SNI) rat model. J Pharmacol Sci 2013;121(1):9–16.
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
38 Hale SL, Shryock JC, Belardinelli L, Sweeney M, Kloner RA. Late sodium current inhibition as a new cardioprotective approach. J Mol Cell Cardiol 2008;44(6):954–67.
50 Shields SD, Cheng X, Uceyler N, et al. Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury. J Neurosci 2012;32(32):10819–32.
Analgesic Effects of Ranolazine Treatment 60 Yoon YW, Na HS, Chung JM. Contributions of injured and intact afferents to neuropathic pain in an experimental rat model. Pain 1996;64(1):27–36. 61 Physicians’ Desk Reference, 67th edition. Montvale NJ: PDR Network LLC; 2013. 62 England JD, Gamboni F, Levinson SR, Finger TE. Changed distribution of sodium channels along demyelinated axons. Proc Natl Acad Sci U S A 1990;87(17):6777–80. 63 Bray GM, Rasminsky M, Aguayo AJ. Interactions between axons and their sheath cells. Annu Rev Neurosci 1981;4:127–62.
65 Jefferys JG. Nonsynaptic modulation of neuronal activity in the brain: Electric currents and extracellular ions. Physiol Rev 1995;75(4):689–723. 66 Black JA, Nikolajsen L, Kroner K, Jensen TS, Waxman SG. Multiple sodium channel isoforms and mitogenactivated protein kinases are present in painful human neuromas. Ann Neurol 2008;64(6):644–53. 67 England JD, Gamboni F, Levinson SR. Increased numbers of sodium channels form along demyelinated axons. Brain Res 1991;548(1–2):334–7. 68 Henry MA, Freking AR, Johnson LR, Levinson SR. Increased sodium channel immunofluorescence at myelinated and demyelinated sites following an inflammatory and partial axotomy lesion of the rat infraorbital nerve. Pain 2006;124(1–2):222–33. 69 Henry MA, Luo S, Foley BD, et al. Sodium channel expression and localization at demyelinated sites in painful human dental pulp. J Pain 2009;10(7): 750–8. 70 Henry MA, Luo S, Levinson SR. Unmyelinated nerve fibers in the human dental pulp express markers for myelinated fibers and show sodium channel accumulations. BMC Neurosci 2012;13:29. doi: 10.1186/ 1471-2202-13-29. 71 Attal N, Bouhassira D. Mechanisms of pain in peripheral neuropathy. Acta Neurol Scand Suppl 1999;173:12–24, discussion 48–52. 72 Decosterd I, Ji RR, Abdi S, Tate S, Woolf CJ. The pattern of expression of the voltage-gated sodium channels Na(v)1.8 and Na(v)1.9 does not change in uninjured primary sensory neurons in experimental neuropathic pain models. Pain 2002;96(3): 269–77.
74 Caldwell JH, Schaller KL, Lasher RS, Peles E, Levinson SR. Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses. Proc Natl Acad Sci U S A 2000;97(10):5616–20. 75 Henry MA, Freking AR, Johnson LR, Levinson SR. Sodium channel Nav1.6 accumulates at the site of infraorbital nerve injury. BMC Neurosci 2007;8:56. 76 Black JA, Frezel N, Dib-Hajj SD, Waxman SG. Expression of Nav1.7 in DRG neurons extends from peripheral terminals in the skin to central preterminal branches and terminals in the dorsal horn. Mol Pain 2012;8:82. doi: 10.1186/1744-8069-8-82. 77 Fried K, Govrin-Lippmann R, Devor M. Close apposition among neighbouring axonal endings in a neuroma. J Neurocytol 1993;22(8):663–81. 78 Lisney SJ, Pover CM. Coupling between fibres involved in sensory nerve neuromata in cats. J Neurol Sci 1983;59(2):255–64. 79 Rasminsky M. Pathophysiology of demyelination. Ann N Y Acad Sci 1984;436:68–85. 80 Rasminsky M. Ectopic generation of impulses and cross-talk in spinal nerve roots of “dystrophic” mice. Ann Neurol 1978;3(4):351–7. 81 Rasminsky M. Ephaptic transmission between single nerve fibres in the spinal nerve roots of dystrophic mice. J Physiol 1980;305:151–69. 82 Rasminsky M. Ectopic excitation, ephaptic excitation and autoexcitation in peripheral nerve fibers of mutant mice. In: Culp W, Ochoa J, eds. Abnormal Nerves and Muscles as Impulse Generators. Oxford: Oxford University Press; 1982:344–62. 83 Rasminsky M. Spontaneous activity and cross-talk in pathological nerve fibers. In: Kandel E, ed. Molecular Neurobiology in Neurology and Psychiatry. New York: Raven Press; 1987:39–49. 84 Seltzer Z, Devor M. Ephaptic transmission in chronically damaged peripheral nerves. Neurology 1979;29(7):1061–4. 85 Sun QQ. A novel role of dendritic gap junction and mechanisms underlying its interaction with thalamocortical conductance in fast spiking inhibitory neurons. BMC Neurosci 2009;10:131. doi: 10.1186/14712202-10-131. 1779
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
64 Dugandzija-Novakovic S, Koszowski AG, Levinson SR, Shrager P. Clustering of Na+ channels and node of Ranvier formation in remyelinating axons. J Neurosci 1995;15(1 Pt 2):492–503.
73 Kretschmer T, Happel LT, England JD, et al. Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry. Acta Neurochir (Wien) 2002;144(8):803–10, discussion 810.
Gould et al. 86 Amaya F, Decosterd I, Samad TA, et al. Diversity of expression of the sensory neuron-specific TTXresistant voltage-gated sodium ion channels SNS and SNS2. Mol Cell Neurosci 2000;15(4):331–42.
91 Bonica J. Anatomic and physiologic basis of nociception and pain. In: Bonica J, ed. The Management of Pain, 2nd edition. Philadelphia: Lea & Febiger; 1990:28–94.
87 Djouhri L, Newton R, Levinson SR, et al. Sensory and electrophysiological properties of guinea-pig sensory neurones expressing Nav 1.7 (PN1) Na+ channel alpha subunit protein. J Physiol 2003;546(Pt 2):565– 76.
92 Harris AL, Spray DC, Bennett MV. Kinetic properties of a voltage-dependent junctional conductance. J Gen Physiol 1981;77(1):95–117.
88 Toledo-Aral JJ, Moss BL, He ZJ, et al. Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons. Proc Natl Acad Sci U S A 1997;94(4):1527–32.
93 Loewenstein WR. Junctional intercellular communication: The cell-to-cell membrane channel. Physiol Rev 1981;61(4):829–913. 94 Spray DC, Harris AL, Bennett MV. Equilibrium properties of a voltage-dependent junctional conductance. J Gen Physiol 1981;77(1):77–93. 95 Bostock H, Sears TA. Continuous conduction in demyelinated mammalian nerve fibers. Nature 1976; 263(5580):786–7.
90 Andrew D, Greenspan JD. Peripheral coding of tonic mechanical cutaneous pain: Comparison of nociceptor activity in rat and human psychophysics. J Neurophysiol 1999;82(5):2641–8.
96 Bostock H, Sears TA. The internodal axon membrane: Electrical excitability and continuous conduction in segmental demyelination. J Physiol 1978;280: 273–301.
1780
Downloaded from http://painmedicine.oxfordjournals.org/ by guest on March 19, 2016
89 Andrew D, Greenspan JD. Mechanical and heat sensitization of cutaneous nociceptors after peripheral inflammation in the rat. J Neurophysiol 1999; 82(5):2649–56.
