Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review

Accepted Manuscript Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review M.K. Hamad, K. He, H.F. Abdulrazeq, A.M. Mustafa, J. Nakhla, M.M. Herzallah, A. Mammis PII:

S1878-8750(18)30159-1

DOI:

10.1016/j.wneu.2018.01.116

Reference:

WNEU 7314

To appear in:

World Neurosurgery

Received Date: 22 August 2017 Revised Date:

15 January 2018

Accepted Date: 16 January 2018

Please cite this article as: Hamad M, He K, Abdulrazeq H, Mustafa A, Nakhla J, Herzallah M, Mammis A, Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review, World Neurosurgery (2018), doi: 10.1016/j.wneu.2018.01.116. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review Department of Neurological Surgery, Rutgers University- New Jersey Medical School

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Hamad MK1, He K1, Abdulrazeq HF1, Mustafa AM2, Nakhla J2, Herzallah MM3, 4, and Mammis A1

1- Department of Neurological Surgery, Rutgers University – New Jersey Medical School 2- Albert Einstein College of Medicine

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3- Center for Molecular and Behavioral Neuroscience, Rutgers University 4- Palestinian Neuroscience Initiative, Al-Quds University

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Disclosures: The authors of this literature review have no conflicts of interest and no disclosures.

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Funding: This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

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Corresponding Author: Mousa K. Hamad

[email protected]

90 Bergen Street, Suite 8100 Newark, NJ, 07101

Attn: Antonios Mammis, MD 973-626-2376

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Abstract: Neuropathic pain is a subset of chronic pain that is caused by neurons that are damaged or firing aberrantly in the peripheral or central nervous systems. The treatment guidelines for neuropathic pain

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include antidepressants, calcium channel α 2 delta ligands, topical therapy, and opioids as a second line option. Unfortunately, pharmacotherapy has not been effective in the treatment of neuropathic pain except in the treatment of trigeminal neuralgia with carbamazepine. The inability to properly treat neuropathic

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pain causes frustration in both the patients and their treating physicians. Venoms, which are classically thought to be causes of pain and death, have peptide components that have been implicated in pain relief.

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Although some venoms are efficacious and have shown benefits in patients, their side effect profile precludes their more widespread use. This review identifies and explores the use of venoms in neuropathic pain relief. This can open doors to potential therapeutic targets. We believe that further research into the mechanisms of action of these receptors as well as their functions in nature will provide

Perspective:

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alternative therapies as well as a window into how they affect neuropathic pain.

Understanding the mechanism of action of these venom toxins can direct future investigation into possible

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therapies for patients with refractory neuropathic pain. Research should be directed at utilizing venoms in

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additional beneficial ways and formulating venoms for the most appropriate pain relief techniques.

Keywords:

Neuropathic pain, venoms, chronic pain, non-opiate analgesia, opioids

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INTRODUCTION Neuropathic pain is a subset of chronic pain that is caused by neurons that are damaged or neurons that are firing aberrantly. It affects 15-19% of the world population, and produces pain that is

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severe enough to interfere with daily life.4, 17, 148 Classically, neuropathic pain is described as a widespread, burning sensation that initiates in seemingly unprovoked waves and is associated with

allodynia and some sensory deficit. Allodynia is described as a sensation where a normally painless

stimulus produces pain. The initial source can be localized to either the peripheral or central nervous

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systems and generally involves pathways connected to nociception.14 Many different disease states

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(diabetes, multiple sclerosis, viral infection, etc.) can lead to neuropathic pain and there are various mechanistic explanations for the onset of pain in each of these diseases. For example, in trigeminal neuralgia, a disease of the trigeminal nerve which causes debilitating attacks of pain in the V1, V2, and V3 dermatomes, the pain may be caused by nerve compression (most commonly by blood vessels) at the site where the peripheral nerve reaches the brainstem. Postherpetic neuralgia on the other hand is caused

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by viral infiltration of nerve roots leading to increased expression of voltage gated sodium and potassium channels, as well as upregulation of receptors involved in pain pathways.56, 134 It is important to consider that various manifestations of neuropathic pain may require different treatment options than those

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currently available.

The search for safe and effective pain control is pertinent now more than ever. Current treatment

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guidelines for neuropathic pain usually start with non-opioid analgesics (such as NSAIDs, acetaminophen, and aspirin) followed by tramadol. Severe non-remitting pain is then treated with opioids, which are classically addictive and can lead to potential abuse. Currently, α2 adrenergic agonists, antidepressants (Tricyclics and SNRIs), and antiepileptics (gabapentin, pregabalin, etc.) are treatment options for opioid-resistant pain.126 These traditional medications have shown limited effectiveness. The addictive potential of opioids and severe side-effect profiles of these drugs when used chronically have also been problematic.

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The rising tide of opiate addiction necessitates new research on pain relief. According to the Centers for Disease Control and Prevention, the amount of prescription opioids sold in the US as well as number of deaths from prescription opioids have quadrupled since 1999. Prescription narcotics continue

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to be widely prescribed, since no effective alternative is available to patients with chronic neuropathic pain.

Venoms, whether they are from snakes, lizards, leeches, snails, scorpions, mammals, or spiders,

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are traditionally sources of severe pain or death. However, analyzing the structure and functions of these venoms in preliminary studies has demonstrated various channel blockers that are more potent and

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effective than current treatment options for neuropathic pain. In this review, the authors have identified a list of toxins that have been used in animal studies or clinical trials with the potential for therapeutic use. The authors have only identified one venom-derived analgesic that is currently in practice for severe intractable neuropathic pain; however, its severe side-effect profile limits its usefulness. An isolated venom peptide, which goes by the generic name Ziconotide, is a synthetic version

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of ω-conotoxin MVIIA derived from the venom of Conus magus, a marine snail. Conotoxins are small peptides derived from the genus Conus, which are all venomous predators. Three different types, α, µ, and ω, interact with a variety of receptors involved in regulating pain. The α-conotoxin interacts with nAchR,

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the ω-conotoxins interacts with both Nav and Cav channels, and the µ-conotoxin targets Nav channels. The ω-conotoxins, from which ziconotide is derived, paralyze prey after VGCCs are inhibited. They have

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been studied extensively for their role in pain relief.65 Ziconotide is currently available as an intrathecal injection after successfully completing three double-blind, placebo-controlled trials and is in use for management of severe chronic pain.65 Although its usefulness as an analgesic is apparent, Ziconotide has an extensive side effect profile including rhabdomyolysis, sleep disturbance, and headache among many others.65, 106 Therefore, with increasing knowledge about options for non-opioid analgesics, it is essential to continue research for safer efficacious alternatives.

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nAChR INHIBITORS

α-conotoxins:

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In reviewing the breadth of research on animal toxins for the treatment of neuropathic pain, the family of conotoxins from which ziconotide is derived comes into sight. Conotoxins, as inhibitors of a variety of ion channels, present as strong candidates in the field of pain relief and as potential alternatives

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to ziconotide because of its adverse side effects and invasive route of administration.65

Sodium channels have been found to play a significant role in the mechanism of pain. After nerve injury, it was found that sodium channels began to accumulate at the site of injury and along the length of

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the axon.122 This resulted in hyperexcitability and spontaneous action potential discharges. Another group also found that nociceptors spontaneously fired after nerve injury.125 A popular but controversial route of analgesia revolves around α9 nicotinic acetylcholine receptors (α9nAChRs) and their relationship with αconotoxins. Although evidence for the involvement of α9nAChRs, and the α9α10 nAChR in particular, in

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pain treatment has been found,97, 164 the types of pain these receptors may be related to as well as their exact mechanism of action have been contested.107, 114 Further research indicates that the putative α9nAChR pain-alleviation mechanism may not require the α9nAChR, but rather incorporate a GABA-

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dependent N-type voltage gated Ca2+ channel pathway;21, 22, 32, 77, 114, 115 recent evidence both supports and precludes the GABA-dependent calcium channel inhibition mechanism of pain relief through α-

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conotoxins.67, 171 As of yet, the true mechanism remains unclear. Three α-conotoxins of interest are the peptides α-conotoxin Vc1.1, α-conotoxin RgIA, and α-conotoxin GeXIVA. 1. α-conotoxin Vc1.1 This toxin, discovered from the mRNA sequence of the Queen Victoria cone sea snail Conus

victoriae,145 has been analyzed for its nAChR selectivity and binding to α9α10.30 Intramuscular injection of α-conotoxin Vc1.1 dose-dependently attenuated mechanical hyperalgesia in chronic constriction injury (CCI) and partial nerve ligation (PNL) models of neuropathic pain.146, 163 The former model calls for exposure and multiple ligations of the sciatic nerve, usually resulting in forms of hyperalgesia and 5

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allodynia. Acute treatment of CCI rats with an intramuscular bolus of α-conotoxin Vc1.1 led to significant attenuation of hyperalgesia, defined by increased weight threshold prior to paw withdrawal. Compared to saline, rats treated with the toxin were able to tolerate up to approximately double the weight before

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withdrawing from pain. This effect lasted between 1 and 3 hours after injection. Short term effects lasted 24 hours after injection and rats who received the maximal dose were restored to pre-operative weight levels before withdrawing. Long term effects yielded significant attenuation of mechanical hyperalgesia

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even at the lowest dose with 50% of attenuation lasting one week after injections.146 Moreover, treatment with substance P illustrated a significantly greater vascular inflammatory response 8 weeks post-injury in

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rats treated with Vc1.1 compared to saline control, suggesting an enhanced recovery of injured neurons. Using normal rats as the control for inflammation at 100% post treatment with substance P, rats treated with Vc1.1 demonstrated up to 83% functional recovery while those treated with saline showed 47%.146 Intramuscular administrations of Vc1.1 have also reduced allodynia in PNL and other neuropathic pain models.77, 188 Intrathecal administration of Vc1.1 dose-dependently reduced mechanical allodynia without

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adverse motor effects as evidenced by rotarod test. Rats treated with the conotoxin had increased paw withdrawal thresholds for up to 6 hours while those treated with saline had no changes in threshold.114 However, other investigators have suggested that the mechanism of conopeptides are dependent on

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pathways other than Vc1.1.171 However, other investigators have suggested that the mechanism of conopeptides are dependent on pathways other than Vc1.1.171 They found that conopeptides had variable

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effects on Vc1.1. in different sensory neurons and that there was only minimal inhibition via this pathway. Further research needs to be done to determine the true pathway of analgesia for conopeptides so that improved therapies may be developed. 2. α-conotoxin RgIA Found in the crown cone sea snail Conus regius,51 this toxin also blocks the α9α10 nAChR, and has been examined for its binding mechanism and stability as a potential drug.7, 51, 52 Intramuscular administrations of RgIA in CCI neuropathic rat models attenuated hyperalgesia and allodynia in a dosedependent manner through chronic and acute treatment, without noted adverse effects. In one study, CCI 6

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rats treated chronically for 14 days with RgIA had progressive decrease in paw withdrawal threshold; 61.7 ± 0.6 g and 65.0 ± 0.9 g in 2 nmol- and 10 nmol-treated group compared to 39.2 ± 1.3 g in the saline treated group.44, 163 Of note is RgIA’s ability to decrease degeneration of both dorsal root ganglion and

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sciatic nerve morphology in the CCI model as well as reduce the glial cell-mediated inflammatory response after intramuscular injection.44 Finally, a recent study using a chemotherapeutic drug-induced (oxaliplatin) model of neuropathic pain revealed dose-dependent reductions in mechanical hyperalgesia

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and mechanical and cold allodynia, alongside prevention of DRG degeneration after intramuscular

injection of RgIA conducted at a set dosage. This study found resolution of pain threshold over the control group value after 21 days of treatment with high dose RgIA. No tolerance to the toxin’s effect was

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noted.129 In the spinal cord, although no significant effects were seen in microglial expression, cotreatment of RgIA and oxaliplatin reduced astrocyte number, measured by glial fibrillary acidic protein (GFAP) expression, compared to oxaliplatin alone.129 Interestingly, RgIA alone increased astrocyte number compared to oxaliplatin alone.129

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3. αO-conotoxin GeXIVA

In addition to the former two toxins, another conotoxin, αO-conotoxin GeXIVA, isolated from the general cone sea snail Conus generalis, was recently identified and studied for its properties.98 Similar

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to Vc1.1 and RgIA, GeXIVA inhibits the α9α10 nAChR and presented significant analgesic effects in the area of neuropathic pain.86, 98 There are three isoforms of GeXIVA: [1,2] (bead form), [1,3] (globular

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form), and [1] (ribbon form), with the [1,2] isomer most potently inhibiting α9α10 nAChR.98 In a CCI rat model, GeXIVa [1,2] produced dose-dependent attenuation of mechanical hyperalgesia comparable to morphine after intramuscular injection, with no significant deficits in motor control as evidenced by a rotarod test.98 An additional study of the [1] isomer also showed significant dose-dependent reduction in mechanical hyperalgesia through intramuscular administration, alongside the slightly better effect of the [1,2] isomer and similar non-significance in the rotarod test.86 The significant effects of [1] only applied to single, acute, short term injection patterns; the pain-relieving effect lost significance by one week after

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treatment periods.86 No motor performance effects were found, nor were any behavior disturbances noted.86 Overall, in spite of the contradicting evidence and uncertainty surrounding the relevancy of

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α9α10 nAChR and GABA-dependent VGCC pathways of pain relief, these α-conotoxins have clear potential in the treatment of neuropathic pain. Future research will likely elucidate the exact mechanism of pain alleviation and the scope of the toxins’ effects, as well as reveal other novel toxins with

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therapeutic implications.

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CbTx and Haditoxin

Two final toxins to note in the family of nAChR inhibitors are cobratoxin (CbTx) and haditoxin. CbTx is believed to inhibit the α7 nAChR as determined by known α7 nAChR agonists and antagonists.58 CbTx, isolated from the Southeast Asian monocled cobra Naja naja kaouthia, is an α-cobratoxin that dose dependently diminished thermal and mechanical hyperalgesia, defined as Thermal Withdrawal Latency

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(TWL) and Mechanical Withdrawal Threshold (MWT) respectively, in a PNL rat model after intrathecal injection. The data show an almost two-fold increase in MWT and a 1.5sec increase in TWL 15 minutes after intrathecal injection of high dose CbTx compared to saline.58 This effect remained significant for

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less than an hour. No information on side effects was provided.58 Additionally, CbTx and a derived analog, receptin, inhibit thermal, arthritic, and chemically-induced inflammatory and abdominal writhing

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pain in animal models.29, 94, 152 Haditoxin, on the other hand, has been shown to have an effect on human nAChRs with a strong preference for α7 nAChR, but no studies have yet been conducted on its applications to neuropathic pain.140

MULTIPLE SITE INHIBITION Bee Venom

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Bee venom, or apitoxin, is a mixture of biologically-active chemicals from the honey bee Apis mellifera. It includes bioamines, phospholipase A2, apamin, melittin, and mast cell degradation peptide.1, 26, 156

Bee venom is a relatively new toxin being studied for its clinical applications; however, bee venom

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therapy has prevailed in traditional Middle Eastern and Eastern medicine for many years and is still in practice today.1, 26

Diluted bee venom (DBV) has been the focus of current research surrounding its use in

acupuncture, known as apipuncture. There are data suggesting DBV’s efficacy as an anti-inflammatory as

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well as an analgesic for neuropathic and non-neuropathic pain.155, 156 A slew of studies have been conducted on subcutaneous DBV apipuncture’s attenuating effect on allodynia, including oxaliplatin-

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induced models of neuropathic pain,75 and spinal nerve ligation models.75, 79, 82, 90, 181, 185 In a spinal cord injury-induced pain rat model, DBV appears to reduce central neuropathic pain, described by an over two-fold increase in mechanical withdrawal threshold and two second increase in thermal withdrawal latency compared to the saline treated group.72 The spinal cord injury model involved spinal cord

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hemisection-induced thermal hyperalgesia and allodynia; after repetitive acupunctural DBV treatment was administered 1-5 days post-surgery, a significantly greater effect in neuropathic pain alleviation was discovered compared to treatment 15-20 days post-surgery and controls.72 This indicates a potential early

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benefit of DBV. Repetitive DBV treatment also produced significantly faster motor function recovery in the spinal cord injury model. In this study, the effect of DBV was noted mostly when it was applied to the

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ipsilateral dorsal root ganglion, suggesting a key role of the dorsal horn in DBV’s mechanism of action.72 Similarly, in a chronic constriction injury rat model, repetitive non-acupuntural subcutaneous DBV injections alleviated thermal hyperalgesia.71 Another study using CCI and subcutaneous apipunctural single injection of DBV reported significant effects on thermal hyperalgesia, but none on mechanical allodynia.138 The mechanism by which bee venom functions appears to involve a complex variety of pathways that is not yet fully understood.25 A proposed mechanism that involves glial cell inhibition cited lack of upregulation of GFAP and Iba-1 as evidence for DBV reduction of glial cell activation.72 Normally, after spinal cord injury, glial scar formation is induced by way of glial cell activation and 9

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recruitment, and biomarkers such as GFAP and Iba-1 are upregulated in the process. Adrenergic, serotonergic, and cholinergic systems have all been implicated.82, 138, 182 Also, in the latter chronic compression injury model, repetitive subcutaneous DBV injection may alleviate pain by way of

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adrenoceptors and increased noradrenergic activity in the locus coeruleus.71 Furthermore, bee venom was presented as a novel method of potentiating the analgesic effects of clonidine, an α2-adrenoreceptor antagonist, in neuropathic thermal hyperalgesia and mechanical

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allodynia.70, 184 Subcutaneous apipuncture of DBV with clonidine attenuated pain comparable to higher doses of clonidine alone and reduced the side effect profile. Rats treated with DBV and clonidine together had a 3.5-fold leftward shift in clonidine’s dose-response curve acutely and a 5-fold leftward shift during

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late phase treatment.184 A similar premise was used in a study comparing DBV with morphine, which displayed stronger and longer lasting effects of cold and mechanical allodynia in the group with combined DBV and morphine administration compared to DBV or morphine alone.75 Although bee venom does pose potential harm to humans due to inherent risks of reactions

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toward bee venom (e.g. anaphylaxis),26 its use in therapy persists. There exist uncontrolled trials on bee venom therapy and prospective studies on its application as alternative medicine.183 However, there are also case reports on instances where a bee sting or bite induced immune thrombocytopenia (ITP)1, 2, 113, 153, Of note is a case report in which the patient presenting with ITP had undergone bee venom

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156, 160

therapy.1 This last report draws attention to a potential complication in bee venom therapy; however,

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given the lack of information on the administration of the therapy in this particular case as well as lack of dosing control during other reports,1, 153, 160 the true benefits and harms are still unclear. Despite the research detailing bee venom’s potential benefit in animal models and in humans, it does cause many of the same pain symptoms that it seeks to treat.26 However, further investigation with improved study models of diluted bee venom and its potential to alleviate neuropathic pain is well warranted.

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Crotoxin Crotoxin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus,

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shows analgesic effects through opioid-independent pathways.190 Muscarinic, serotonergic, and adrenergic receptor antagonists all inhibited the anti-hyperalgesic effect of directly applied crotoxin on sciatic nerve transection rats.121 However, studies have provided contradicting evidence regarding the involvement of the cholinergic system with varying results dependent on the dosage of anticholinergic

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chemicals.121, 190 Direct application of crotoxin to transected branches of sciatic nerve in rats immediately after surgery also showed a long lasting (64 days) reduction in hyperalgesia, whereas subcutaneous

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administration only produced a short term alleviation of pain (between 2 and 24 hours).121 No adverse effects on motor activity were noted after either direct application or intraperitoneal crotoxin injection.121, 169

Further evidence of crotoxin’s potential use in analgesia comes from an early Phase I clinical trial

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for cancer treatment in which the toxin was administered intramuscularly for the treatment of solid tumors.33 Although pain relief was not a primary endpoint of the experiment, a majority (18/23) of the evaluated patients experienced a decrease in cancer-related pain illustrated by a reduction in analgesic use

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and patient input.33

VOLTAGE-GATED CALCIUM CHANNEL INHIBITORS CVIE and VIF

CVIE and CVIF are conotoxins isolated from a cDNA library of the Conus catus. They act by

inhibiting N-type VGCCs. The peptides were found to reduce allodynic behavior in the rat PNL model.11 They inhibited the Cav2.2 channel in a reversible and voltage-dependent manner in the dorsal horn of the spinal cord. CVIE in particular was found to have the highest potency in inhibiting N-type VGCCs and was also able to partially reverse impaired weight bearing in rats when given subcutaneously.141 No major 11

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side effects when administering the conotoxins systemically were observed. Due to the potency and lack of side effects seen thus far in these studies, CVIE seems a promising candidate for the treatment of neuropathic pain. However, it is important to note the limitation of the data as studies have only been

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conducted in rat models and CVIE and CVIF have not been studied pharmacologically in humans.

Phα1β

Phα1β is a toxin isolated from the venom of the Brazilian wandering spider Phoneutria

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nigriventer. It reversibly inhibits N-type VGCCs.162 It has only recently been studied for its potent effects

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on the nervous system in various forms of pain and pruritus in animal models.36, 37, 101 Not only does Phα1β appear to possess a broad range of applications in pain management, but it also shows promise over ziconotide in treatment of neuropathic, inflammatory, post-operative, visceral, and cancer pain in relation to strength and duration of analgesia and mitigation of side effect profile in some but not all pain models.24, 36, 37, 46, 135, 136

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Its proposed mechanism of action implicates the N-type VGCC, which has an important role in the production of pain as evidenced by ziconitide. Typically, VGCCs are activated upon receipt of depolarizing stimulus, causing an intracellular cascade that results in exocytotic release of vesicular

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compounds in the synaptic terminal.132 The N-type channel itself is associated with neuronal pain induction,132 exhibiting colocalization with substance P and inhibition through a µ-opioid receptor

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signaling pathway.150, 168

In the management of neuropathic pain, Phα1β shows a significant benefit in chronic constrictive

injury rat models and chemotherapeutic-induced models of neuropathic pain.37, 135, 139 In one study, rats were either intrathecally injected with a single dose of Phα1β 8 days post injection, or continuously administered the toxin for 7 days against a control.139 Results showed significant reduction in CCIinduced neuropathic pain in both cases, with the single injection attenuating pain by up to 100% for 1-6 hours and the continuous administration for 1-7 days by 63% ± 13%.139 In the paclitaxel-induced chronic neuropathic pain model, single intrathecal injection of Phα1β significantly attenuated mechanical 12

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hyperalgesia in a dose-dependent manner for 4 hours after long term administration of paclitaxel. In the acute pain stage, rats treated with Phα1β showed a maximum inhibition of mechanical pain of 100%, and those in the chronic phase had a maximum inhibition of 81 ± 10% with no statistically significant effect

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on control rats.135 Importantly, no significant behavioral side effects ensued from neither continuous treatment with Phα1β nor low analgesic doses;135, 139 dynamic allodynia significantly presented in the highest analgesic dose of Phα1β, but the comparative side effect of ziconotide was greater.135 The

Other Toxins of Phoneutria nigriventer

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harmful side effects associated with ziconotide.37, 135, 139

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intrathecal administration of Phα1β displays similar if not greater efficacy than ziconotide, without the

The venom of Phoneutria nigriventer also contains several less-studied toxins with potential pain-alleviating properties. In a mechanistic overview of these toxins, δ-Ctenitoxin-Pn1a, formerly known as PnTx4(6-1), binds to insect axonal voltage-gated sodium channels (VGSCs) to prolong the action

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potential via delay of Na+ channel inactivation, but does not have an effect on the electrophysiology of mammalian Na+ channels.35 PnTx4(5-5) is another toxin from Phoneutria nigriventer that is cited to possess analgesic properties;53 however, aside from one of its recombinant forms having an effect on

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increasing depolarization threshold of VGSCs, there has been little research on its relationship with pain.53, 130 Likewise, rPnTx1 acts as a VGSC inhibitor and competes with µ-conotoxin for binding sites,

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but has not yet been studied for pain treatment.102, 154 The toxin Tx3-3 inhibit P/Q, R, and L-type VGCCs (P/Q and R more effectively) and the toxin Tx3-5 inhibits L-type channels.23, 53, 81 The toxins from Phoneutria nigrivente have demonstrated the ability for global inhibition of VGCCs, which is a promising component in understanding pain inhibition.132 Among the aforementioned toxins, δ-CNTX-Pn1a, Tx3-3, and Tx3-5 all indicate potential for neuropathic pain treatment, albeit only via intrathecal administration.34, 53, 127 δ-CNTX-Pn1a produced significant effects on neuropathic rat models with induced CCI, showing brief (20 min) attenuation of hyperalgesia compared to control, although there was no mention of side effects.53 Similarly, in PNL and 13

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streptozotocin-induced diabetic neuropathy models, Tx3-3 produced significant alleviation of allodynia with no adverse motor effects commonly associated with VGCC blockade.34 Lastly, administration of Tx3-5 in another PNL model produced a maximum inhibition of mechanical hyperalgesia of

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87±10%, with no noted adverse effects. This study also tested Tx3-5 on a model of cancer-related pain with morphine tolerance in mice showing complete reversal of melanoma-related mechanical

hyperalgesia.127 Much like Phα1β, these toxins contributed to other areas of pain treatment, including

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largely untapped potential for treatment in pain management.

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inflammatory and post-operational pain.53, 127 As a whole, the toxins of Phoneutria nigriventer present a

SNX-482

SNX-482 is a peptide toxin isolated from the giant baboon spider Hysterocrates gigas and a blocker of a variety of calcium, sodium, and potassium channels;6, 57, 76, 116 it has been used to study the VGCC Cav2.3 in particular.76 Although its putative significance to neuropathic pain lies in its ability to

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block this R-type VGCC, which has been shown to contribute at least in part to pain sensation in inflammatory models,109 recent research comparing wild type mice to Cav2.3 knockouts illustrate no significant difference in the development of neuropathic pain in the form of partial sciatic nerve ligation-

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induced mechanical allodynia in mice.177 Regardless, the use of SNX-482 in a spinal nerve ligation rat model of neuropathic pain reduced sensitivity to thermal and mechanical allodynia and issued a dose-

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dependent reduction of C and Ad-fiber nociceptor responses after intrathecal administration,103 indicating a potential for further neuropathic pain research.

VOLTAGE-GATED SODIUM CHANNEL INHIBITORS Tetrodotoxin In addition to VGCCs, the literature shows VGSCs play a significant role in pain transmission.31, 137

The significance of VGSCs was alluded to with the mechanisms of PnTx4(5-5) and PnTx4(6-1). The 14

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premier animal-derived blocker of VGSCs is the puffer fish venom tetrodotoxin (TTX). A non-peptide, its applications fell under the scrutiny of the scientific community early on,158 and it has accumulated a large wealth of research to support its potential as an analgesic. Analysis and use of the venom in an array of

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experiments has elucidated not only its mechanism of action but the importance of VGSCs in pain.137 VGSCs have even been characterized into TTX-sensitive (Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.6, Nav1.7) and TTX-resistant (Nav1.5, Nav1.8, Nav1.9) depending on the concentration of TTX required to

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block the channel. This has allowed for a variety of studies on the comparative importance and effects of TTX-sensitive and TTX-resistant subtypes of VGSCs in pain. Channels Nav1.3, Nav1.7, Nav1.8, and

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Nav1.9 have all been implicated in contributing to neuropathic pain.137 However, there exists conflicting evidence from knockout studies that exhibit normal neuropathic pain development in the absence of such channels.137

As for the direct effects of TTX on neuropathic pain, early studies have illustrated its ability to dose-dependently reduce abnormal neuronal activity after sciatic nerve transection through an intravenous

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route of administration and mechanical sensitivity in a spinal nerve ligation (SNL) model of neuropathic pain.99, 128 In the latter study, routes of administration were also compared; intraperitoneal injection of TTX did not significantly reduce mechanical allodynia compared to control, but epidural and direct

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administration to the dorsal root ganglion did have a significant effect.99 Although no side effects were mentioned, the concentration of TTX used in the study was below that necessary to block action

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potentials.99 In a chemotherapy-induced neuropathic pain mouse model, subcutaneous TTX was shown to attenuate allodynia and thermal hyperalgesia without adverse side effects, with an additional preventative effect on allodynia pre-administration of chemotherapeutic agent.118 Intramuscular TTX additionally displayed antihyperalgesic effects without observable motor impairment for treatment of neuropathic muscle pain induced by another chemotherapeutic agent.5 The effect was time-dependent, with TTX administration 4 hours after oxaliplatin injection producing stronger analgesic effects than administration 15 days after oxaliplatin injection.5 The analgesic effect lasted longer in the 4 hours post-oxaliplatin injection group as well, remaining significant after 2 hours.5 In another model of neuropathic pain, CCI of 15

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the sciatic and the infraorbital nerve produced mechanical hyperalgesia and allodynia that was attenuated at an optimal dose of 3 µg/kg of subcutaneous TTX in both acute and long term treatment patterns.74 At this and higher dosages of TTX there were no observable adverse effects and no impaired motor

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performance as evidenced by a rotarod test. This more recent study also indicated that its effect in CCIsciatic nerve rats may involve interaction with the opioid pathway; interestingly, antagonization of the opioid pathway appears to augment TTX’s effects.74 For a comprehensive review of TTX and its impact

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on neuropathic and other forms of pain, refer to Nieto et al’s review article.119

Importantly, TTX has already moved through a significant amount of clinical testing. Randomized, double-blind, placebo-controlled studies and longitudinal studies have been conducted on

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patients with cancer pain.61, 62 For example, one study demonstrated a clinically important use that TTX may provide in the form of analgesia for patients who have persistent moderate to severe cancer pain despite best analgesic care. This study has shown that the mean duration of analgesic response to TTX was 56.7 days compared to 9.9 days for the placebo. Some adverse effects were associated with TTX

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including nausea, dizziness, and oral numbness or tingling, however, there were generally mild to moderate and transient.63 Most common adverse events were nausea, dizziness, and oral numbness or tingling and were generally mild to moderate and transient. Overall, the effects have been promising with

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a number of patients reporting analgesic effects, although further study is needed. In addition to completion of Phase III cancer pain trials, TTX has already completed Phase II clinical trials for

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chemotherapy-induced neuropathic pain and is proceeding to Phase III. Thus, of the potential animal toxins and venoms for neuropathic pain management discussed herein, tetrodotoxin is currently the closest to realization.

ProTx-II VGSC Nav1.7 is of particular interest due to evidence regarding its significance in regulating neuropathic pain, alongside a range of human pain disorders.45, 137, 180 Loss of function mutations, for instance, in the gene encoding Nav1.7 can result in diminished pain perception in humans.45 16

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ProTx-II, a peptide of the inhibitory cysteine knot family isolated from venom of the tarantula Thrixopelma pruriens, binds with strong affinity to voltage-gating domains on sodium channels and Nav1.7 in particular, inhibiting the channel through increase of voltage activation threshold.147, 174, 175

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Recent evidence has shown that the toxin may do so through a lipid membrane binding mechanism, in which hydrophobic residues interact with the lipid membrane to position the toxin for interaction with its binding site on the channel.66 Its 100-fold selectivity for Nav1.7 over other channels makes it a likely target for development of analgesics; indeed, it has been tested for its ability to regulate acute

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inflammatory pain and neuropathic pain.147, 159 Intravenous ProTx-II failed to show any significant

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increase in paw withdrawal threshold in rats subjected to injections of Complete Freund’s Adjuvant (CFA) for a model of acute inflammatory pain. These rats presented with mechanical allodynia, which was slightly but insignificantly attenuated by the toxin.147 The toxin found more success in alleviating diabetes-induced thermal hyperalgesia in a neuropathic pain mouse model. Following diabetic induction via intravenous injection of streptozotocin, intrathecal administration of increasing doses of ProTx-II

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(0.04 – 4 ng) resulted in a dose-dependent relationship with tail flick latency, indicating an increase in pain threshold that peaked 60 minutes after administration and subsided by 180 minutes.159 This implicates the Nav1.7 channel with thermal hyperalgesia in diabetic mice. However, there was no

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mention of side effects, and western blotting illustrated no significant difference in dorsal root ganglion Nav1.7 channels in diabetic versus non-diabetic mice. As damage to the nervous system in neuropathic

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pain has been shown to change expression and regulation of voltage-gated sodium channels,137 the full picture of the interaction between Nav1.7 and other sodium channels and pain is, once again, not fully understood. This leaves room for further analysis of ProTx-II and other VGSC-blocking toxins for insight into neuropathic pain.

µ-theraphotoxin-Hhn1b (HNTX-IV) and µ-theraphotoxin-Hs2a (HWTX-IV) Two families of tarantula toxins, hainantoxins and huwentoxins, present additional potential for use in therapeutic treatment of neuropathic pain. µ-TRTX-Hhn1b (HNTX-IV) is a peptide isolated from 17

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the venom of Haplopelma hainanum and HWTX-IV is a peptide isolated from the venom of Haplopelma schmidti.170 Much like ProTx-II and other spider toxins,15, 78 HNTX-IV20, 172 and HWTX-IV39, 95, 173, 174 act as inhibitors of the TTX-sensitive VGSC Nav1.7, among others.

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HNTX-IV significantly reduced neuropathic pain in a rat model.92 The study utilized a spared nerve injury (SNI) model of neuropathic pain. An alternative to CCI, SNI calls for exposure of the sciatic nerve and its three endings but ligation of only two out of the three endings.38 SNI-induced mechanical

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allodynia underwent dose-dependent attenuation following intraperitoneal injection of HNTX-IV. The pain-alleviating effect peaked at approximately 60 minutes and lasted beyond 100 minutes with the

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highest dose of 0.2 mg/kg, in contrast with a dose of 40 mg/kg of mexiletine, a VGSC blocker positive control whose effect became insignificant at 60 minutes. Side effects including inactivity and paralysis were evident in rats given high doses (0.2 mg/kg) of HNTX-IV shown by rotarod.92 HWTX-IV dose dependently produced a longer and more potent pain alleviating effect on allodynia than mexiletine in a spinal nerve model.93 Moreover, HNTX-IV and HWTX-IV both produced significant dose-dependent

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effects on hyperalgesia in inflammatory rat models induced by acetic acid and formalin at a potency comparable to morphine. Rats with spinal nerve injury-induced pain treated with HNTX-IV had a significant increase in withdrawal threshold, 8.8 ± 0.9 g, compared to saline treated rats, 1.4 ± 0.4 g.92, 93

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Overall, these two peptides are candidates for further analysis of their effects on alleviating neuropathic pain.

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As there are other hainantoxins85, 96, 176 and huwentoxins165, 166 that show selective inhibition of VGSCs, future research warrants investigation into these and other spider toxins as potential therapeutics.142

µ-SLPTX-Ssm6a Other ion channel blockers include a variety of centipede venoms, whose analgesic and antimicrobial properties pose potential therapeutic benefits.64, 161 Several venoms have been isolated from the Chinese red-headed centipede Scolopendra subspinipes mutilans,178 one of which has a significant 18

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effect in relieving pain.179 µ-SLPTX-Ssm6a, a potent and highly selective inhibitor of human Nav1.7, significantly alleviates pain in chemically-induced models of inflammatory pain and thermal pain after intraperitoneal injection without notable adverse effects. This effect was observed in a dose dependent

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relationship and was comparable to morphine in the thermal pain and acetic acid-induced pain models, being even more potent than morphine in the formalin-induced inflammatory pain model.179 However, other studies have shown that the µ-SLPTX-Ssm6a peptide is not a strong binder of Nav1.7, which calls into question the true analgesic mechanism.111 Although µ-SLPTX-Ssm6a has not yet been tested in

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neuropathic pain models, its selectivity, relative stability,179 and potential analgesic properties make it a

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promising therapeutic candidate and likely subject of future experimentation.

GpTx-1

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Venom of Grammostola porteri, the Chilean tarantula, contains GpTx-1. Study of its analogues illustrate an ability to inhibit Nav1.7.110-112 In the only study of its effect on pain, intraplantar coadministration of GpTx-1 with OD1, a scorpion-derived toxin exhibiting delayed inactivation of

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Nav1.7, significantly reduced spontaneous pain behavior characterized by control OD1 in a dosedependent manner.40 However, this novel pain model has not been characterized as neuropathic, and

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GpTx-1 failed to produce an effect when injected intraperitoneally at 0.1 mg/kg. Higher doses could not be administered as they resulted in significant motor impairment.40

Anntoxin

The first gene-coded amphibian neurotoxin, anntoxin was isolated from the tree frog Hyla annectans and was shown to inhibit TTX-sensitive VGSCs.186 Anntoxin has antinociceptive effects in models of inflammatory and thermal pain, through a potential anti-inflammatory mechanism.167 Although

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no experiments on animal models of neuropathic pain have yet been conducted, anntoxin is a new

ACID-SENSING ION CHANNEL INHIBITORS PcTx1 and Mambalgins

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candidate that should be examined in future research.

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A more recently studied player in the field of pain is the acid-sensing ion channel (ASIC), whose isoforms (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) associate to form homomeric and heteromeric combinations of cation channels in peripheral and central neurons.43 Several studies have

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arisen to implicate ASICs in the treatment of various forms of pain,43, 105 including postoperative,42 acidic, and primary inflammatory pains.41 Animal toxin agonists of ASIC channels even demonstrate a paininducing effect.13 This evidence has created an impetus for further investigations into its pharmacological benefits.105

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In the context of neuropathic pain, the realm of ASIC inhibitors provides two candidates: PcTx1, a peptide toxin isolated from the venom of Psalmopoeus cambridgei,170 and the mambalgins, a series of peptides (1-3) found in the venom of the black mamba Dendroaspis polylepis.48 Both of these toxins have

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been investigated as ASIC inhibitors to examine their potential benefit to therapeutic analgesia.9 The former, PcTx1, was the first inhibitor of ASIC to show high affinity and selectivity for the channel,

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inhibiting ASIC1a and heteromeric combinations of ASIC1a/ASIC2a and ASIC1a/ASIC2b.54, 69 The peptide’s structure and character enable highly sensitive interactions with ASICs,28, 143 and the toxin inhibits ASIC1a through a desensitization mechanism that involves increasing its H+ affinity.27 Intrathecal injection of PcTx1 significantly reduced thermal hyperalgesia and mechanical allodynia in a CCI neuropathic mouse model and thermal and mechanical hyperalgesia in a vincristine (anticancer drug associated with neuropathy) rat model.104, 123 The effects were comparable to morphine and persisted at an hour after injection. The peptide is neither lethal nor causative of secondary effects associated with morphine. As for a potential mechanism of action, a model of thermally-induced acute pain was used to 20

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elucidate a reliance on the endogenous opioids enkephalins as well as their associated receptors, the µand δ-opioid receptors, with the ASIC1a channel as a potential intermediary.104 However, as the mechanistic findings were not presented in a model of neuropathic pain, it is difficult to assert likewise

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association of the opioid pathway. These findings present alongside additional comparable analgesic effects to morphine in the areas of thermally and chemically-induced acute and inflammatory pain.104 The ASIC inhibitors mambalgins 1 and 2 have shown promise in the treatment of neuropathic pain and have undergone research in analyzing their binding to ASIC1a as well as in developing an

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efficient synthetic procedure.48, 108, 131, 144, 149 Intravenous and intrathecal injections of mambalgin-1

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demonstrated significant attenuation of mechanical and thermal hyperalgesia in a CCI mouse model.49 The hyperalgesia-reducing effect of intravenous mambalgin-1 diminished earlier than that of intrathecal administration and results indicated that the mechanism of intravenous mambalgin-1 is independent of both ASIC1a and the opioid pathway. Both ASIC1a-KO mice and those pretreated with naloxone, an opioid antagonist, showed no respective significant differences from treatment with mambalgin-1 alone,

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alluding to a mechanistic pathway involving other types of ASICs. However, intrathecal administration of mambalgin-1 produced longer-lasting effects than intravenous mambalgin-1 but revealed naloxonesensitivity as well as total lack of function in ASIC1a-KO mice. No apparent toxicity or motor problems

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were observed with the mambalgin-treated mice.49 In addition to indicating the analgesic potential o fmambalgin-1, these results also suggest a significant variability in ASIC expression and role in different

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locations and in different pain pathways, elucidating an underexplored area for further research into ASICS in transmission of neuropathic and other forms of pain. APETx2 is another notable ASIC inhibitor that comes from Anthropleura elegantissima, a sea

anemone. This inhibitor blocks both ASIC3 and TTX-R Nav1.8 channels,12, 47 and evidence shows dose dependent attenuation of pain through intramuscular and intraplantar routes of injection in acid-induced muscle pain and CFA-induced inflammatory pain models, respectively.73 No studies on APETx2 and its action on neuropathic pain have yet been conducted.

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MECHANOSENSITIVE ION CHANNEL INHIBITOR

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GsMTx4 GsMTx4, a toxin from the Chilean rose tarantula Grammostola rosea, is the only known inhibitor of mechanosensitive ion channels.16 In a therapeutic context, these channels have mainly been studied for

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their involvement in conditions such as cardiac arrhythmias and muscular dystrophy.10, 16, 157 Despite results indicating that GsMTx4 is ineffective in reducing mechanically-activated currents in cultured DRG neurons expressing stretch-activated ion channels, analgesic effects have been evidenced in vivo and

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implicate the stretch activated ion channels TRPV4, TRPC1, and TRPC6.3, 50, 133 The resulting decrease in mechanical hyperalgesia was only tested using TRPV4-deficient mouse models that had pain induced by inflammatory soup.3 Research has revealed this toxin to be effective in reducing mechanical allodynia in a sciatic nerve injury rat model through intraperitoneal injection, indicating a potential inhibition of

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mechanosensitive ion channels.133 Moreover, in a paclitaxel chemotherapy-induced neuropathic pain model, intradermal GsMTx4 reversed mechanical hyperalgesia after single injection, but this effect did not persist after 24 hours.3 It is important to note that GsMTx4 has activity on a variety of channels

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involved in pain (Kv and Piezo1) which may contribute to its analgesic effects.8, 120 Future research on this toxin and its ability to induce analgesia in the context of mechanosensitive receptors should be

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explored.

OPIOID PATHWAY Crotalphine

Crotalphine, a small 14-amino acid long peptide, is a venom isolated from Crotalus durissus terrificus that has been shown to provide significant analgesic effects in models of inflammatory, neuropathic, and cancer pain.19, 59, 60, 80 Recent studies of crotalphine have primarily focused on deducing 22

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the pathway by which the toxin takes its effect. These studies have implicated a joint cannabinoid-opioid pathway, in which peripheral pain is alleviated through activation of the CB2 cannabinoid receptor by crotalphine and release of endogenous opioid A-dynorphin, which acts on the κ-opioid receptor.100, 187

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Additionally, crotalphine was found to desensitize TRPA1 channels and reduced chemically-induced inflammatory hyperalgesia in mouse models.18 This desensitizing effect disappeared in TRPA-1 deficient mice. These mechanistic results were obtained through models of inflammatory pain, although κ and δ-

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opioid receptor antagonists did inhibit crotalphine’s effect in CCI rats.60 In terms of its effect on

neuropathic pain itself, CCI rat models illustrated crotalphine’s analgesic effect in dose dependently

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reducing mechanical hyperalgesia and mechanical allodynia after oral administration for more than 3 days. Pain threshold was increased to above pre-surgery levels in tests of mechanical hyperalgesia, and crotalphine proved to be more effective than both morphine and gabapentin in alleviating pain.59 A similar effect was found in CCI rats, in which both intraplantar and oral crotalphine significantly reduced mechanical hyperalgesia and allodynia.60 Overall, there was no significant effect on motor activity after

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oral administration of crotalphine.59 Moreover, oral administration of crotalphine every 3 days for 75 days and evaluating its analgesic effect 60 minutes after each administration did not reveal any tolerance development.59 This suggests a need for further studies investigating its uses in neuropathic pain.

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The ability of this isolated animal toxin to decrease neuropathic pain even when administered orally makes it unique. Stability to this degree has not been previously observed from a peptide toxin

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given orally, and the mechanism by which crotalphine functions when administered orally has not been determined.80 Non-central routes of administration that effectively alleviate neuropathic pain provide new opportunities to advance future pain therapies.

Najanalgesin Pain reduction resulting from the najanalgesin toxin peptide, isolated from the venom of the Chinese cobra Naja naja atra, demonstrates a distinctive mechanism of action.68 The toxin may produce its analgesic effects through a glial cell related pathway reminiscent of diluted bee venom, but it had 23

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initially been shown to involve the opioid and cholinergic pathways.68, 87, 88, 91 Recent evidence of najanalgesin toxin peptide’s mechanism of action indicates that it may occur through the c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) pathway, one of the several MAPK pathways activated

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in response to nerve damage.55, 89 Intrathecal and intraperitoneal injections of najanalgesin produced significant antinociceptive effects in an SNL rat model.87, 88 The intraperitoneal experiments showed a dose dependent analgesic effect on mechanical allodynia lasting for over 10 days after a single injection,

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which persisted longer than the effects of morphine. The reduction on thermal allodynia, however, was not dose dependent. Intrathecal injections of najanalgesin produced significant dose dependent alleviation of mechanical allodynia, but for a shorter duration than intraperitoneal injection; this effect was not

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compared to morphine.88 Intrathecally administered najanalgesin also reduced SNL-induced hyperalgesia.87 Continued research on najanalgesin and its exact mechanism of action could further

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support its candidacy as a target of neuropathic pain treatment.

NOREPINEPHRINE TRANSPORTER INHIBITOR Xen2174

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Isolated from the marbled cone, Conus marmoreus, Xen2174 is a more stable analog of the cconotoxin MrIA. It is a noncompetitive inhibitor of the norepinephrine transporter (NET) and thus affects

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the noradrenergic pathway.151 Importantly, it was shown to have potential in neuropathic pain treatment through intrathecal injections in CCI rat models. Xen2174 dose dependently alleviated mechanical allodynia with mild side effects and had a longer duration of effect than morphine.117 These and other findings have propelled Xen2174 into clinical trials for postoperative and cancer pain, and there is promise for its use in neuropathic pain.84, 124 However, Xen2174 has halted its clinical development in 2012. The invasive administration of Xen2174 for examining its therapeutic effects further complicates its use in clinical trials.

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DISCUSSION Numerous potential avenues for isolated toxin peptides exist for alleviating neuropathic pain but

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require further exploration in terms of safety and efficacy (see supplementary Table 1). There are common themes among the toxin peptides in terms of mechanism of action with many concentrating on VGSCs (see supplementary Table 2) and VGCCs (see supplementary Table 3).83, 189 This is expected,

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as ziconotide, currently the only synthetically produced and FDA-approved prescription toxin peptide for pain relief, is a VGCC inhibitor. Nav1.1-1.19 are VGSCs believed to contribute to neuropathic pain.

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However, the data has not been pharmacologically tested in humans, which limits the ability to draw conclusions regarding their role in neuropathic pain. In the literature, Nav1.7 stands out as the foremost target of the VGSC inhibitors. All the VGSC-inhibiting toxins discussed act on TTX-sensitive VGSCs, and the toxins TTX, ProTx-II, HNTX-IV, HWTX-IV, µ-SLPTX-Ssm6a, and GpTx-1 all possess painreducing effects that may arise through their high selectivity for Nav1.7. Although the only FDA-

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approved animal toxin for neuropathic pain management is the VGCC inhibitor ziconotide, the research on other VGCC inhibitors is surprisingly less focused than that on sodium channels. Spider venoms SNX482, Pha1b, Tx3-3, and Tx5-5 act on VGCCs to produce their cited effects on neuropathic pain, but only

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when administered intrathecally. Unfortunately, intrathecal injection proves far too invasive to be done routinely and an alternative procedure or route of administration would need to be sought out for these

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toxin peptides. If a more favorable method of introducing the peptide could be demonstrated, then it appears that the isolated spider venom peptides would have an improved side effect profile when compared to other frequently used drugs. Though these toxin peptides hold promise in treating chronic pain, few of these molecules have been formally tested on humans and not all underwent testing for side effects in animal models (e.g. rotarod test, open field test). There exists high variability in the pathways that could be used to alleviate neuropathic pain. In addition to action upon the classic VGCCs, VGSCs, and opioid receptor-mediated pathways, the potential candidacies of nAChRs and NETs (inhibited by α-conotoxins Vc1.1/RgIA and c-conotoxin MrIA, 25

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respectively) in neuropathic pain sensation warrants a re-evaluation of these pathways. In fact, recent findings have established ASICs and MSICs as putative modulators of neuropathic pain sensation. Neuropathic pain is understood as a complex phenomenon that likely has as many methods of alleviation

field of venoms and toxins that may be examined in the future.

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as sources of induction. The scope of species touched upon in this review can attest to the unexplored

The copious number of pathways through which neuropathic pain can be relieved is further

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augmented by the variety of administration methods recognized in rat models. Although the majority of treatments showing attenuation of neuropathic pain occur through the intrathecal route, some studies

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show success using less invasive procedures. The α-conotoxins Vc1.1 and RgIA as well as the aOconotoxins GeXIVA [1,2] and [1,4] produce their effects through intramuscular administration; the blowfish toxin TTX reduced abnormal neuronal activity intravenously and pain subcutaneously and intramuscularly; DBV’s effects were observed using the subcutaneous method; the tarantula toxin HNTXIV was intraperitoneally injected; mambalgin-1 was intravenously administered; the tarantula toxin

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GsMTx1 was effective through both intraperitoneal and intradermal injections; the cobra toxin najanalgesin was intraperitoneal; crotoxin was subcutaneously administered; and GpTx-1 and crotalphine were both effective after intraplantar injection. Crotalphine’s stability imbued it with effectiveness

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through an oral route of administration in spite of its status as a peptide, the only tested toxin peptide to demonstrate this unique characteristic. The significance of this finding should not be lost on the ongoing

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search for a less invasive method of neuropathic pain relief, a leap to the ideal route of drug administration. Nonetheless, the other methods of injection remain improvements on the central route. Further research on administration methods, as well as on prolonging the stability of these toxins, is needed.

CONCLUSION Overall, it seems that there is a high potential among the venoms and toxin peptides discussed 26

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herein for a few to be developed into therapeutic entities. Many show positive effects in significantly attenuating or reversing neuropathic pain in an array of animal models but need further testing. Researchers in these experiments employ a variety of administration methods, which show promise in

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improving treatment procedures that move away from invasive practices. Adverse side effects were either not observed or limited for a large portion of the tested compounds. More intensive research into these toxins’ impact on animals, as well as optimization of administration methods and side effect profiles, may

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likely advance some compounds to consideration for clinical trials. Neuropathic pain, albeit a complex and largely invisible inducer of suffering, is a condition that has the potential to be fought with the advent

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of new and more effective drugs derived from naturally occurring venoms.

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Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review Highlights

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Drugs derived from naturally occurring toxins may aid in treating neuropathic pain. Researching toxins’ impact on animals may lead to consideration for clinical trials. Ziconotide is an available toxin drug used for management of severe chronic pain. Many toxin peptides have only undergone animal studies. It is essential to continue research for safer alternatives to opioid analgesia.

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Abbreviation List: ASIC - Acid-sensing ion channel Ca – Calcium CCI - chronic constriction injury cDNA - complementary Deoxyribonucleic acid CFA - Complete Freund’s Adjuvant DBV - Diluted bee venom DRG – Dorsal Root Ganglion FDA – Food and Drug Administration GABA - gamma-Aminobutyric acid GFAP - glial fibrillary acidic protein HNTX-IV - μ-TRTX-Hhn1b ITP - induced immune thrombocytopenia KO – Knock out MAPK - mitogen-activated protein kinase MWT - Mechanical Withdrawal Threshold MSIC – Mechanosensitive Ion Channels mRNA – Messenger Ribonucleic acid Na – Sodium nAChR – Nicotinic Acetylcholine Receptor NET - norepinephrine transporter NSAID – Nonsteroidal Anti-Inflammatory Drug PNL - partial nerve ligation SNI - spared nerve injury SNL - spinal nerve ligation SNRI –Serotonin Norepinephrine Reuptake Inhibitor TTX - Tetrodotoxin TWL - Thermal Withdrawal Latency US – United States V1 – Ophthalmic nerve V2 – Maxillary Nerve V3 – Mandibular nerve VGCC – Voltage gated calcium channel VGSCs - voltage-gated sodium channels μg/kg – Microgram per kilogram

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August 12, 2017

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Mousa K Hamad Albert Einstein College of Medicine Department of Neurosurgery 1300 Morris Park Ave Bronx, NY 1046 [email protected]

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Thank you for your consideration.

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Very Respectfully, Mousa K Hamad, MD Corresponding Author Albert Einstein College of Medicine Department of Neurosurgery