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Upregulation of voltage-gated sodium channels (VGSCs) and Na+/K+-ATPase (sodium pumps) is common across most malignant carcinomas. Targeted osmotic lysis (TOL) is a developing technology in which the concomitant stimulation of VGSCs and pharmacological blockade of sodium pumps causes rapid selective osmotic lysis of carcinoma cells. This treatment of cervical carcinoma is evidence that TOL is a safe, well-tolerated and effective treatment for aggressive advanced carcinomas that has the potential to extend life without compromising its quality. TOL is likely to have broad application for the treatment of advanced-stage carcinomas.

// Harry J. Gould III 1, 3, 5, 6, 7 , Jack Norleans 2 , T. David Ward 4 , Chasiti Reid 2 and Dennis Paul 1, 2, 3, 5, 6, 7 1 Department of Neurology, Louisiana State University Health Sciences Center, New Orleans, LA, USA 2 Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA, USA 3 Department of Anesthesiology, Louisiana State University Health Sciences Center, New Orleans, LA, USA 4 Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, New Orleans, LA, USA 5 Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA 6 Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, LA, USA 7 Center of Excellence for Oral and Craniofacial Biology, Louisiana State University Health Sciences Center, New Orleans, LA, USA Correspondence to: Harry J. Gould III, email: hgould@lsuhsc.edu Dennis Paul, email: dpaul@lsuhsc.edu Keywords: targeted osmotic lysis; epithelial carcinoma; cancer; sodium channels; sodium pumps Received: October 29, 2017 Accepted: February 21, 2018 Epub: February 26, 2018 Published: March 20, 2018 ABSTRACT Sodium influx through voltage-gated sodium channels (VGSCs) coupled with balanced removal of sodium ions via Na + , K + -ATPase is a major determinant of cellular homeostasis and intracellular ionic concentration. Interestingly, many metastatic carcinomas express high levels of these channels. We hypothesized that if excess VGSCs are activated and Na + , K + -ATPase is simultaneously blocked, the intracellular Na + concentration should increase, resulting in water movement into the cell, causing swelling and lytic cell death. MDA-MB-231 breast cancer cells over-express VGSCs by 7-fold. To test our hypothesis, we treated these cells in vitro with the Na + , K + -ATPase blocker, ouabain, and then stimulated with a sublethal electric current. For in vivo histologic and survival studies, MDA-MB-231 xenografts were established in Nu/J mice. Mice injected with saline or ouabain were electrically stimulated with trains of 10 msec 10V DC pulses. Within seconds to minutes, the cells swelled and lysed. MCF-10a cells, which express normal VGSCs levels, were unaffected by this treatment. Cells from the weakly-malignant cell line, MCF-7, which express 3-fold greater VGSCs than MCF-10a cells, displayed an intermediate time-to-lysis. The rate of lysis correlated directly with the degree of sodium channel expression and malignancy. We also demonstrated efficacy in cell lines from prostate, colon and lung carcinomas. Treated MDA-MB-231 xenografts showed 60–80% cell death. In survival studies, TOL-treated mice showed significantly slower tumor growth vs. controls. These results are evidence that this ”targeted osmotic lysis” represents a novel method for selectively killing cancer cells and warrants further investigation as a potential treatment for advanced and end-stage breast cancer.

Changes in membrane potential dramatically impact the progression of cancer, yet little is known as to how ion transport contributes to disease progression. Voltage-gated sodium channels (VGSCs) and Na,K-ATPases are particularly attractive targets as both are upregulated in many cancers and have been correlated specifically with invasiveness in vitro and metastasis and in vivo. However, their precise upstream regulators and downstream effectors remain unclear. Additionally, it has not been determined whether these transporters are co-regulated in cancer, although they display overlap in their downstream effectors and have complementary activity on sodium ions. Such a system has, however, been described in neuronal models of pain, wherein inflammatory injury leads to an upregulation in VGSC expression and a subsequent proportional increase in Na,K-ATPase. We sought to describe a similar phenomenon in cancer cells, which could indicate that this neuronal response to injury may be hijacked as a driver of metastatic development. To elucidate the impact of inflammatory mediators on these ion transporters in cancer cells, we assessed the effects of TNFα and PGE2 on VGSCs and Na,K-ATPases in the human breast epithelial or cancer cells MCF-10a, MCF-7, and MDA-MB-231. RT-qPCR revealed increases in RNA expression for the clinically relevant VGSC subunit Nav1.5. Similar RNA increases were observed in the α1 and α3 subunits of Na,K-ATPase, although in all cases the magnitude and time point of maximal effect differed by cell line. Then a 1hr challenge of TNFα on MDA-MB-231 cells was used to examine whether the targets were independently regulated by inflammatory mediators, co-regulated via the transport of sodium ions, or co-regulated by the expression of one protein influencing the expression of the other protein. Transfection with siRNA against either the α1 or α3 subunit of Na,K-ATPase did not inhibit the inflammatory-mediated increase in RNA for Nav1.5. However, transfection with anti-Nav1.5 siRNA inhibited the upregulation of both Na,K-ATPase subunits. TNFα challenge following pre-incubation of MDA-MB-231 cells in a sodium-free medium completely inhibited the inflammatory-mediated upregulation of Nav1.5, Na,K-ATPase α1 or α3 RNA. Co-incubation of TNFα with the VGSC blocking drug protoxin I inhibited the inflammatory-mediated increase in Na,K-ATPase α1 or α3 RNA, but co-incubation with the Na,K-ATPase blocking drug ouabain did not inhibit the increase in Nav1.5 RNA and indeed may have potentiated the increase. Together these results are evidence that VGSCs and Na,K-ATPase are co-regulated by inflammatory mediators in metastatic breast cancer, that sodium is essential to this mechanism, and that the presence and ion-transporting function of VGSCs is necessary for the inflammatory-mediated effect on Na,K-ATPase but not vice versa. Citation Format: Steven D. Scahill, Harry J. Gould, Dennis Paul. Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 276.

Voltage-gated sodium channels (VGSCs) are upregulated in aggressive carcinomas, making these channels a target for novel clinical therapies. Targeted Osmotic Lysis (TOL) is a novel cancer therapy that combines pharmacologically blocking Na+,K+, ATPase (sodium pumps) while stimulating VGSCs with a pulsed electric field. The consequent increase in intracellular Na+ causes an osmotic lysis of the cells that overexpress VGSCs. Noncancerous cells, with fewer VGSCs do not lyse. We hypothesized that variability in efficacy of TOL is due to VGSC expression that varies across the cell cycle. We assessed cell surface expression of VGSCs and Na+,K+-ATPase during phases of the cell cycle in which there are single copies of DNA compared with phases in which DNA has doubled. For this, DNA was labeled with either propidium iodide or DAPI and VGSCs were labeled with a pan-specific VGSC antibody while Na+,K+-ATPases were labelled with a pan-specific Na+,K+-ATPase antibody in eight immortalized cancer and non-cancer cell lines. With flow cytometry we showed that VGSC expression doubled during phases of cell division (S-M phases) while Na+,K+-ATPase expression increased by 1.5-fold. To further elucidate the role of VGSCs and Na+,K+-ATPases throughout the cell cycle, S-Trityl-L-cysteine (STLC) was used to suspend the cell cycle in M phase. We hypothesized that treating cancer cells with TOL in the S-M phases would lead to increased cell death, compared to cells that were not suspended in M phase by STLC (G0-G1 phases). Cells were either treated with STLC or media without drug for 24 hours, then exposed to TOL treatment. The efficacy of the treatment was measured using a MTT assay revealing that cell death was greater in STLC-treated cells than control cells. By using cell cycle inhibitors, we may increase the efficacy of TOL for treating advanced carcinomas that overexpress VGSCs. Citation Format: Samantha Edenfield, Harry J. Gould, Dennis Paul. Efficacy of targeted osmotic lysis is increased by cell cycle inhibition in M Phase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2312.

In vitro cancer research predominantly utilizes established cell lines of a chosen cancer type as phenotypic representations of various disease states, whether they be non-cancerous, non-invasive, highly metastatic, or derived from distinct cellular origins within an organ or tissue. While such methodology is useful to study cancer cells from diverse disease states, when one considers the tremendous complexity of intertumoral heterogeneity, it becomes problematic to use cell lines derived from distinct individuals with diverse malignancies to study the progression of tumorigenesis in a single cancer type. To address this need, a novel murine model was developed, composed of six cell lines isolated from progressively advanced tumors all derived from a single parental line (Ray et al., Exp. Cell Res. 340, 1-11). Each cell line should represent a snapshot of a particular stage of this cancer’s development, with the entire model providing a platform to study tumorigenic changes incurred as the transformed cells progressed from a localized tumor to an aggressively malignant growth. However, isolating primary cell lines can be an inefficient process, and a successfully isolated line may not accurately represent the dominant neoplastic cell type from a tumor. For that reason, it is necessary to characterize the cell lines from this model to confirm that the observed in vitro behavior of the cells matches the expected phenotype of the tissue from which it was derived. To this end, we performed an in vitro characterization of each cell line in the model. The three lines derived from distant metastatic tumors displayed significantly higher rates of two dimensional motility and invasive capacity as compared to the parental cell line, the line derived from a primary tumor or from a local metastasis. The distant metastatic lines also displayed elevated rates of extracellular acidification, an indication of increased rates of aerobic glycolysis. These measures demonstrated convincingly that the behavior of the cells in culture mirrored the expected phenotype of the tissue from which they were derived, thus validating the utility of the model. We then performed a comprehensive proteomic analysis via orbitrap tandem mass spectrometry, yielding expression data on over 3,000 proteins across all cell lines. This analysis yielded expected results that further validated the model, such as showing an increasing trend of expression of cancer-associated proteins like transcription factor AP-1 and decreasing trends of expression of tumor suppressors like protein NDRG1. However, it also revealed striking changes in protein expression in targets not currently implicated in tumorigenesis, thus providing novel targets for further examination. In total, this study validated a novel murine cell model as a valuable tool in the study of tumorigenesis, while providing a robust proteomic data set to guide future research into cancerous progression. Citation Format: Steven D. Scahill, Kelly Jean Sherman, Jessie Guidry, Harry J. Gould, Dennis Paul. In vitro characterization of a novel murine model of cancerous progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1684.

Targeted osmotic lysis (TOL), the concurrent stimulation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps, kills up to 100% of highly malignant, human and murine breast cancer cells in vitro and slows the growth of murine breast cancer homografts and increases survival of the hosts in vivo when compared to homografts treated with negative controls. In this study we compared the efficacy of TOL and paclitaxel, a current standard chemotherapeutic agent for treating advanced murine breast cancer, as a positive control. For this, BALB/c mice with 4T1 homografts were treated with TOL alone, paclitaxel alone or with a combination regimen of paclitaxel and TOL. For TOL, mice received 8 hourly doses (3 mg/kg) of digoxin on 2 successive days. Upon reaching steady-state levels (dose 5) the mice were exposed to 4 X 30 minutes of pulsed electric field stimulation (18 V/m, 10 ms pulses, 15 interstimulus intervals) hourly for a total of 2 hours of stimulation. Alternatively, mice received paclitaxel (20 mg/kg) by i.p. injection. Mice that were treated concurrently with TOL and paclitaxel received paclitaxel 2 days prior to treatment with TOL. Groups of mice that received digoxin alone, stimulation alone, paclitaxel + digoxin, paclitaxel + stimulation or vehicle alone served as negative controls. Initially, the homografts were measured daily and then every other day with a digital caliper until criteria for humane endpoint euthanasia were met. The mean time to meeting criteria for humane endpoint euthanasia in mice treated with paclitaxel alone (2 days) was identical to that observed in the negative controls. Mice treated with TOL alone, on average, met criteria at post-treatment day 5, whereas administration of paclitaxel two days before TOL reduced the effectiveness of TOL, decreasing mean survival to day 3 post-treatment. Immunohistochemical evidence indicated that paclitaxel’s effect on tubulin may impede the transport and insertion of channels into the cell membrane, thereby reducing the channel density and the effectiveness of TOL. By contrast, the administration of TOL either before or after treatment with paclitaxel improved host survival. Normal tissues were unaffected. These results are evidence that TOL is equal to or better than paclitaxel for increasing survival of murine hosts with advanced breast cancer when TOL is administered before or between paclitaxel dosing cycles. Sequential use of TOL may well improve survival, but simultaneous administration of paclitaxel and TOL will likely reduce TOL’s adjunctive benefit. Because of the conserved nature of the sodium channel/sodium pump mechanism, the comparable efficacy to TOL to a currently approved treatment and the favorable adverse effect profile, we submit that TOL warrants further study as a promising option for treating individuals with advanced neoplastic disease. Citation Format: Harry J. Gould, Kelly J. Sherman, Dennis Paul. Efficacy of targeted osmotic lysis using pulsed electric field stimulation compared to paclitaxel for treating murine, triple-negative breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1071.

Voltage-gated sodium channels (VGSCs) are the target for many therapies. Variation in membrane potential occurs throughout the cell cycle, yet little attention has been devoted to VGSCs and Na+,K+-ATPase in the cell cycle. We hypothesized that in addition to doubling DNA and cell membrane in anticipation of cell division, there should be a doubling of VGSCs and Na+,K+-ATPase compared to non-dividing cells. We tested this hypothesis in eight immortalized cell lines by correlating immunocytofluorescent labeling of VGSCs or Na+,K+-ATPase, with propidium iodide or DAPI fluorescence using flow cytometry. Cell surface expression of VGSCs during phases S through M was double that seen during phases G0 - G1. By contrast, Na+,K+-ATPase expression increased only 1.5-fold. The increases were independent of baseline expression of channels or pumps. The variation in VGSC and Na+,K+-ATPase expression has implications for both our understanding of sodium's role in controlling the cell cycle and variability of treatments targeted at these components of the Na+ handling system.

Concurrent activation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps causes a targeted osmotic lysis (TOL) of carcinomas that over-express the VGSCs. Unfortunately, electrical current bypasses tumors or tumor sections because of the variable resistance of the extracellular microenvironment. This study assesses pulsed magnetic fields (PMFs) as a potential source for activating VGSCs to initiate TOL in vitro and in vivo as PMFs are unaffected by nonconductive tissues. In vitro, PMFs (0&ndash, 80 mT, 10 msec pulses, 15 pps for 10 min) combined with digoxin-lysed (500 nM) MDA-MB-231 breast cancer cells stimulus-dependently. Untreated, stimulation-only, and digoxin-only control cells did not lyse. MCF-10a normal breast cells were also unaffected. MDA-MB-231 cells did not lyse in a Na+-free buffer. In vivo, 30 min of PMF stimulation of MDA-MB-231 xenografts in J/Nu mice or 4T1 homografts in BALB/c mice, concurrently treated with 7 mg/kg digoxin reduced tumor size by 60&ndash, 100%. Kidney, spleen, skin and muscle from these animals were unaffected. Stimulation-only and digoxin-only controls were similar to untreated tumors. BALB/C mice with 4T1 homografts survived significantly longer than mice in the three control groups. The data presented is evidence that the PMFs to activate VGSCs in TOL provide sufficient energy to lyse highly malignant cells in vitro and to reduce tumor growth of highly malignant grafts and improve host survival in vivo, thus supporting targeted osmotic lysis of cancer as a possible method for treating late-stage carcinomas without compromising noncancerous tissues.

Chronic pain is a major factor that impairs functionality and compromises quality of life. Unfortunately, this type of pain is often under-treated due to lack of education about the use and range of effective non-pharmacologic modalities, about how to use and monitor pharmacologic modalities, and the fear about real and imagined adverse effects that are associated with its management. Cancer is diagnosed in approximately 40% of the population with up to 89% experiencing some form of pain in the later stages of the disease. Unfortunately, a significant portion of this population receives inadequate treatment for their pain. Here we provide a review of the evidence-base for determining the best approach for managing the pain of malignancy in the hope of providing a basic framework for the physician to better utilize the pharmacological options that comprise an important component of comprehensive pain care for the cancer patient. This review contains 2 figures, 7 tables, and 106 references. Keywords: Cancer pain, malignant pain, opioids, chronic pain, analgesic options, adjuvant options, cancer pharmacology, evidence-based pain treatment

In years past, the prevailing approach to providing pain control was focused on identifying underlying etiologies or pathologic syndromes, e.g., low back pain, trigeminal neuralgia, and cancer pain, that produce the pain. While treating the presumed source of the pain, attempts to improve the accompanying discomfort relied largely on the use of non-opioid medications and the limited use of opioid and adjuvant analgesics. Over the past 25 years, however, there has been a dramatic increase in our understanding of the nervous system and how stimuli associated with actual or potential tissue injury are transduced, transmitted, modulated, perceived, and interpreted to form the basis for initiating appropriate evasive or protective behavior, thereby avoiding or limiting injury. Our current bank of knowledge has led to the recognition that (1) pain in the chronic state is in itself a disease deserving consideration, assessment, and management; (2) pain is not a single entity but a complex, multifaceted experience that warrants detailed and comprehensive evaluation to elucidate symptoms that may reflect specific associated mechanisms amenable to targeted treatment (Woolf and Decosterd, Pain 6(Suppl), S141–7, 1999; Woolf and Max, Anesthesiology 95, 241–9, 2001); and (3) treatment modalities and management approaches not heretofore considered can be effective and can improve the quality of life for those suffering with pain. This chapter will provide a brief overview of the anatomy of pain that forms the basis for current practice.

Opioid analgesics are currently the most effective pharmacologic option for the management of both acute and chronic forms of moderate-to-severe pain. Although the "as-needed" use of immediate-release formulations is considered optimum for treating acute, painful episodes of limited duration, the scheduled dosing of extended-release formulations with immediate-release supplementation for breakthrough pain is regarded to be most effective for managing chronic conditions requiring around-the-clock treatment. The recent introduction of extended-release formulations of the opioid analgesic hydrocodone potentially broadened the possibility of providing pain relief for individuals for whom current formulations are either ineffective or not tolerated. However, reaction to the approval of the new formulations has fueled controversy over the general safety and need for opioid medications, in light of their potential for misuse, abuse, diversion, and addiction. Here, we discuss how the approval of extended-release formulations of hydrocodone and the emotionally charged controversy over their release may affect physician prescribing and the care available to patients in need of chronic opioid therapy for the management of pain.

Pain is a distinct, multidimensional sensory experience that is a common clinical complaint but is essential for survival. Our current bank of knowledge has led to the recognition that (1) pain in the chronic state is in itself a disease deserving consideration, assessment, and management, (2) pain is not a single entity but a complex, multifaceted experience that warrants detailed and comprehensive evaluation to elucidate symptoms that may reflect specific associated mechanisms amenable to targeted treatment (Woolf and Decosterd, 1999; Woolf and Max, 2001), and (3) treatment modalities and management approaches not heretofore considered can be effective and can improve the quality of life for those suffering with pain. This article describes the characteristics of acute and chronic pain, the specialized neural elements in the periphery, spinal cord, and brain that subserve or modulate pain, the roles of various neurochemicals, and the changes induced by damage or disease. The goal of this article is to improve the understanding of the pathways, neurochemistry, transmitters and their relationship to disease or symptomatology, in order to improve treatment and enhance knowledge of future providers.

Sodium channels play a pivotal role in maintaining homeostasis and proper intracellular ion concentrations that are vital to all living cells for function and survival. In excitable tissues such as neurons and heart cells, sodium channels are responsible for establishing and maintaining the transmembrane electrochemical gradient, which is critical for intercellular communication and for the transduction, generation, modulation, and transmission of impulses that underlie normal physiologic function, the perception of stimuli, and the execution of appropriate behavioral responses. Injury and disease often affect changes in the channel density and subtype distribution present in cellular membranes, thereby upsetting the critical electrochemical balance necessary for normal functioning. When such changes affect the systems that process noxious stimulation and are acute and transient, they are beneficial. The pain that is perceived alerts us to current or impending injury and aids in vigilance during wound healing. But when the changes are persistent, the painful signals are no longer protective but, instead, become unrelenting and destructive to the quality of life. Because changes in sodium channel quantity and distribution play such a central role in the perception of pain and the development and maintenance of nociceptive chronicity, significant effort has been expended on discovering ways to affect sodium channel expression and function that might be effective in preventing or managing many painful conditions. The implications of modifying sodium channel expression and function for future therapeutic benefit are the subject of this review. Key Words: Acute pain, chronic pain, sodium channels

Primary headache is a common malady that is often under-recognized and frequently inadequately managed in spite of the fact that it affects up to 95 % of the population in a lifetime. Many forms of headache, including episodic tension and migraine headaches, if properly diagnosed, are reasonably amenable to treatment, but a smaller, though not insignificant, percent of the population suffer daily from a chronic, intractable form of headache that destroys one's productivity and quality of life. These patients are frequently seen in neurological practices at a point when treatment options are limited and largely ineffective. In the following review, we will discuss mechanisms drawn from recent studies that address the transition from acute to chronic pain that may apply to the transformation from episodic to chronic daily headaches which may offer opportunities for preempting headache transformation.

Inadequately assessed and poorly managed pain contributes both to high general medical cost and to increasing numbers of societal problems. A large determining factor of inadequate pain care is the lack of understanding about the complex nature of pain and of a rational approach to its assessment and treatment. The less than adequate assessment of neuropathic pain and its relatively poor response to typical analgesic medications are major determinants to the undertreatment of pain. Other than the important management concept of treating the underlying cause, current approaches to the treatment of pain in general, and neuropathic pain in particular, have shifted away from treatment of individual syndromes toward the identification and management of common symptoms and the mechanisms upon which such symptoms are presumed to be based. This article summarizes the features of neuropathic pain that commonly appear in most peripheral neuropathies, regardless of the mechanism of injury, and provides an approach for the selection of treatment.

Dear Editor We were pleased to learn about the observations of Wu et al. ⇓ related to the effect of ranolazine on doxorubicin-induced changes in the late sodium current that occur in dorsal root …

Objective: Although transient neurologic symptoms may occur after neuraxial anesthetics with lidocaine, permanent neurologic symptoms (PNS) are unusual after uncomplicated continuous epidural analgesia (CEA). This report demonstrates that PNS may follow CEA uncomplicated by paresthesias, dural puncture, spinal hemorrhage, or intravascular local anesthetic injection; and performed with bupivacaine alone administered at high dosages with vasoconstrictors over a prolonged time period. Case Report: An obstetrical patient developed fixed left L5-S1 sensorimotor radiculopathy following L5-S1 level CEA with bupivacaine alone administered at high doses (270 mg) with a vasoconstrictor (epinephrine, 225 mg) over a prolonged time period (62 hours). Conclusions: CEA should be administered at higher lumbar levels for labor analgesia rather than at lumbosacral levels that may require higher volumes and concentrations of local anesthetics with greater potential for local anesthetic neurotoxicity. Local anesthetics with vasoconstrictors should only be used as sentinel markers of unrecognized intravascular entry, rather than as primary local anesthetics during CEA to avoid any potential for ischemic neuropathy.

Neuropathic pain is a common and difficult to manage public health problem characterized by frequent treatment failure and high management costs. The variable presentation and response to treatment among patients make it difficult for physicians to apply a single, standardized approach for management. The physician's role in treating neuropathic pain is complex. Clinical decisions must be drawn from personal and shared experience, case reports, and evidence-based, controlled trials performed on selected populations of patients with common, narrowly-defined conditions. Recent advances in our understanding of the mechanisms that underlie the processing of nociceptive stimuli and the perception of pain have led to the increased ‘off-label use’ of adjuvant medications in an attempt to provide relief for many patients who heretofore have suffered unnecessarily with intractable neuropathic pain. Unfortunately, as with any treatment, sound clinical decisions can occasionally result in an untoward adverse response. It is therefore imperative that potential adverse effects inherent to all medications be considered and weighed against the untoward consequences of withholding treatment prior to incorporating their use in any course of management. This commentary presents a case report that illustrates a particularly devastating consequence that was encountered when a medication was selected for ‘off-label use’ in the treatment of intractable pain and presents an opinion for consideration in developing guidelines for determining acceptable risk and standard of care based upon rational adherence or deviation from the approved indications offered for the use of a medication at the time of its introduction into practice is granted.

Recently, the U.S. Food and Drug Administration (FDA) approved Zohydro(®), an extended release formulation of the opioid analgesic hydrocodone that contains no acetaminophen. This approval was against the recommendation of the FDA's Expert Panel. Subsequently, both chronic pain advocates and anti-drug abuse advocates have steadfastly expressed their support of, or astonishment at this decision. Here, we review the pharmacokinetics, pharmacodynamics, safety and abuse liability of this hydrocodone formulation and how it relates to the Expert Panel's opinion and the FDA decision. We discuss the important issues, risk mitigation, potential use of abuse deterrents, and how the different viewpoints of the Expert Panel and FDA decision makers resulted in the approval and subsequent controversy.

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. 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 voltage-gated 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 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. Conclusions Ranolazine exerts broad-spectrum actions to reduce mechanical allodynia that is associated with peripheral demyelination injury.

Inflammation causes upregulation of NaV1.7 sodium channels in the associated dorsal root ganglia (DRG). The resultant increase in sodium influx must be countered to maintain osmotic homeostasis. The primary mechanism to pump sodium out of neurons is Na(+), K(+)-ATPase. To test whether there is a compensatory upregulation of Na(+), K(+)-ATPase after inflammation, rats received an injection of complete Freund's adjuvant (CFA) into one hindpaw and saline into the contralateral hindpaw. Three days later, L4-L6 DRGs were extracted and analyzed using gel electrophoresis and immunohistochemistry. Immunoreactivity for both the α-1 and α-3 subunits were increased in DRG associated with CFA-treatment, compared to saline-treatment. To test whether dysregulation of Na(+), K(+)-ATPase may cause cell death after inflammation, we produced a pharmacological blockade with ouabain (10mg/kg, s.c.) three days after CFA injection and paws were stimulated or not. Twenty-four hours later, DRG were removed and stained with cresyl violet. Greater cell death was seen in DRG from ouabain-treated animals on the CFA treated side than the saline-treated side. Paw stimulation doubled this difference. Control DRG showed little neuronal death. These results are evidence that regulation of Na(+), K(+)-ATPase during major inflammatory disease states is critical for homeostatic protection of primary afferent neurons.

The development of thermal allodynia in relationship to sodium channel augmentation in dorsal root ganglia (DRGs) was studied in albino rats. Paw withdrawal latencies were measured hourly following complete Freund's adjuvant (CFA) injections. Sodium channels were demonstrated with immunocytochemistry. Sustained minimum latencies were attained between 10 and 12 h post-injection. Sodium channel labeling began to increase at 23 h post-injection and reached maximum levels by 24 h. Thermal hypersensitivity is thus established 12 h before sodium channel augmentation can be demonstrated.

The mechanisms by which inflammation induces a chronic pain state are poorly understood. Following the induction of many painful conditions, an increase in the spontaneous firing rate of neurons is often observed in peripheral sensory ganglia. Since ion channels are essential mediators of spike generation and impulse conduction, it is reasonable to postulate that local changes in ion channel expression might underlie the changes in membrane excitability. Such alterations may serve to enhance the efficiency by which painful stimuli are transduced and then conducted to the central nervous system. In these studies, we employed immunocytochemical methods to investigate the changes in sodium channel expression in dorsal root ganglia of rats following a subcutaneous injection of complete Freund's adjuvant, an inducer of chronic inflammation. We find that sodium channel immunoreactivity within primary sensory neurons is dramatically increased within 24 h of the complete Freund's adjuvant injection. These changes persist in small neurons for at least 2 months and roughly parallel the time course of behaviorally measured changes in pain thresholds. Thus, the regulation of sodium channel synthesis may play a role in the generation and maintenance of the hyperesthetic state seen in chronic inflammation.

Opioid medications are frequently used in the effective treatment of intractable pain, but prolonged use of such medications can be complicated by a host of adverse effects. Among these adverse effects, tolerance and mental clouding can be especially disabling and can lead to both a reduced effectiveness of treatment and a reduced quality of life for many requiring treatment with these medications. Here we examined the relative contributions of complete Freund's adjuvant (CFA)-induced inflammatory pain and opioid medication on spatial memory for a well-learned task in male Sprague-Dawley rats. Although CFA, by itself, had little effect on spatial memory, morphine administered to pain-free animals produced profound detrimental effects on spatial memory that persisted longer than the analgesic effectiveness of the drug. However, no significant cognitive deficits were observed in animals receiving morphine in the presence of CFA-induced pain. Taken together, these results are evidence that the pain state of the organism can alter some of the negative effects of morphine.

Anti-epilepsy medications have frequently beenused in the treatment of neuropathic pain to reduce thetransmission of nociceptive signals. 1,2 Zonisamide is a sulfona-mide drug that was approved in 2000 for the adjunctive treat- ment of partial seizures in adults. Because of the unique com-bination of mechanisms through which zonisamide functions,it has potential to be a useful option in the treatment of re-fractory neuropathic pain. We present the case of a patientwith refractory neuropathic pain related to idiopathic poly-neuropathy, who responded to adjunctive treatment withzonisamide. Key Words: anticonvulsants, sulfonamides, adjunctivetherapy CASE REPORT A 53-year-old female presented for evaluation of chronicpain associated with idiopathic sensorimotor neurop-athy. Her symptoms began in 1992 with constant burn-ing in her toes that gradually intensified and extendedproximally to her knees. The pain was lancinating inquality, was exacerbated by light touch and heat, andwas associated with numbness in a stocking distribu-tion. Her pain intensity was rated an average of 9 out of10 on a visual analog scale (VAS), where 0 is no pain,and 10 is the worst pain imaginable. Her past medicalhistory was positive only for hypertension. Prior to pre-sentation, she had undergone extensive diagnostic test-ing for peripheral neuropathy, which included screeningfor diabetes, HIV, ethanol, Lyme disease, connective tis-sue disorders, occult malignancy, and heavy metals, allof which were negative. She was found to have a mildlylow vitamin B

In years past, the prevailing approach to providing pain control was focused on identifying underlying etiologies or pathologic syndromes, e.g., low back pain, trigeminal neuralgia, and cancer pain, that produce the pain. While treating the presumed source of the pain, attempts to improve the accompanying discomfort relied largely on the use of non-opioid medications and the limited use of opioid and adjuvant analgesics. Over the past 25 years, however, there has been a dramatic increase in our understanding of the nervous system and how stimuli associated with actual or potential tissue injury are transduced, transmitted, modulated, perceived, and interpreted to form the basis for initiating appropriate evasive or protective behavior, thereby avoiding or limiting injury. Our current bank of knowledge has led to the recognition that (1) pain in the chronic state is in itself a disease deserving consideration, assessment, and management, (2) pain is not a single entity but a complex, multifaceted experience that warrants detailed and comprehensive evaluation to elucidate symptoms that may reflect specific associated mechanisms amenable to targeted treatment [1, 2], and (3) treatment modalities and management approaches not heretofore considered can be effective and can improve the quality of life for those suffering with pain. This chapter will provide a brief overview of the anatomy of pain that forms the basis for current practice.

Objective. To compare the sensitivity of stimulating the plantar and dorsal hindpaw surfaces in the detection of mechanical allodynia and morphine analgesia. Background. Several approaches are used to assess nociceptive reactivity to mechanical stimulation in animal models of pain. Although certain techniques seem to be favored for studying specific nociceptive conditions, the differences between techniques have not been directly compared and characterized. We chose to compare methods employing stimulation applied to the dorsum of the paw with stimulation of the plantar surface to demonstrate the utility of each approach in determining baseline nociceptive thresholds, changes in those thresholds after injury, and analgesic efficacy. Methods. Withdrawal thresholds from mechanical stimulation applied to the dorsal and plantar surface of the hindpaw were measured in rats treated with morphine after receiving subcutaneous injections of complete Freund's adjuvant (CFA) using Semmes-Weinstein (S-W) monofilaments and electro von Frey (EVF) stimulation. Results. In contrast to stimulation of the dorsal surface, plantar hindpaw stimulation seldom elicited an aversive withdrawal response. Differences in withdrawal response from baseline were only detectible within the first 5 hours post-CFA and only with EVF stimulation. No significant differences in stimulation techniques were observed after the initial 5-hour window. Effective dose 50 (ED50) for analgesic efficacy was consistently lower using dorsal stimulation. Conclusions. Stimulation of the plantar surface of the paw is superior for detecting small changes in paw sensitivity at very low stimulus intensities, whereas stimulation of the dorsal surface is superior for delineating baseline pain thresholds and for detecting robust analgesia. Clinical Relevance. Reliable and sensitive assessment of animal pain behaviors is critical to translational pain research. This study demonstrates the importance of using proper test protocols in animal studies and its implication in preclinical screening of potential analgesics.

Objective. To determine the effect of streptozotocin (STZ)-induced diabetes on the development and recovery of thermal and mechanical hyperalgesia associated with inflammation induced by subcutaneous injection of complete Freund's adjuvant (CFA). Background. The response to nociceptive injury in diabetes differs from that seen in normal individuals in that diabetic patients have increased susceptibility to infections and recover slowly or incompletely from infections and tissue injury due to an abnormal inflammatory response. We have chosen to examine the effect of STZ-induced hypoinsulinemia on the hyperalgesia associated with the enhanced inflammatory state that is induced by the subcutaneous injection of CFA to delineate the potential role of insulin in the development of chronic pain. Methods. STZ- and vehicle-treated Sprague-Dawley rats were tested using thermal and mechanical stimulation after subcutaneous injection of CFA. The behavioral response was compared with that similarly determined in non-diabetic controls and insulin-depleted rats that received insulin replacement. Results. Recovery of the thermal hyperalgesic response to baseline levels occurred over a period of 9–14 days, but the allodynic response to mechanical stimulation persisted for the duration of the study in STZ-treated rats. Insulin replacement prevented the delay in recovery of mechanical allodynia, but had no obvious effect on nociception in uninflamed tissue. Conclusions. Normal insulin function is essential for recovery from mechanical allodynia associated with inflammation induced by CFA. Altered insulin metabolism may selectively influence fiber-type specific mechanisms related to mechanical allodynia associated with inflammation and wound healing.