The Use of Neurotoxins for Palliative Treatment of Chronic Joint Pain

Osteoarthritis is a significant public health problem and is rapidly increasing in prevalence with the aging population. Pain is the most disabling consequence of osteoarthritis. Treatment for pain is inadequate and needs to be addressed with new therapeutic modalities. Chronic pain is often the result of peripheral and central pain sensitization which reduces the pain threshold and increases the perceived pain response to noxious and even non-noxious stimuli. Neurotoxins can reduce this sensitization by various mechanisms and are a fertile area of research for the treatment of chronic pain. Botulinum toxins, vanilloids, and conotoxin have all been studied for the treatment of chronic pain. Botulinum toxins and vanilloids have the greatest potential as analgesics for chronic joint pain thus far. Monoclonal antibodies directed against nerve growth factor have also been developed for the treatment of chronic joint pain due to osteoarthritis. These antibodies are not technically neurotoxins but have significant analgesic potential. However, they may have undesirable side effects and are still being evaluated as possible therapies for chronic osteoarthritis pain.


Introduction
Chronic joint pain is a world-wide problem. Osteoarthritis (OA) is the most common cause of chronic joint pain and is increasing in prevalence. In the United States alone, osteoarthritis affects approximately 27 million adults and is expected to exceed 67 million by the year 2030 [1,2]. Although disability due to osteoarthritis varies, pain is the most disabling symptom affecting OA patients [3,4]. To date, there are no disease modifying treatments for osteoarthritis. Treatment goals are focused on relief of symptoms and minimizing disability. The direct costs of treatment of OA combined with the indirect costs due to lost wages are substantial. According to one estimate, this cost accounts for 2% of the annual gross domestic product [5,6]. Management of chronic pain from OA is challenging. Non-pharmacologic options include education, exercise, weight reduction, acupuncture, and joint protection, but these practices are generally insufficient to provide joint pain relief. Pharmacologic options include systemic and intraarticular therapies [7]. Insufficient pain relief, intolerable drug side effects and drug interactions increase the risk-benefit ratio for available pharmaceutical therapies [8]. Even surgical therapies for degenerative joint disease may not be effective. Knee joint lavage has been shown to be no more effective for alleviation of pain than placebo [9,10].
In particular, end stage disease provides challenges for effective therapy. Opioids can sometimes be effective when other therapies have lost efficacy, but the use of narcotics for chronic pain is undesirable due to eventual dependence and loss of efficacy, the need for dose escalation to maintain effectiveness, and the rising problem of opioid abuse and overdose that has occurred since the use of longacting narcotics have been available [11]. In addition, unacceptable side effects, particularly in the elderly who are more likely to have end stage osteoarthritis pain, makes the use of opioids a poor choice [12]. Finally, opioids may not have any increased efficacy for chronic OA pain compared with non-narcotic therapies [13]. The efficacy of intra-articular treatments such as corticosteroids and hyaluronic acid preparations have not been clearly demonstrated. For this reason the American Academy of Orthopedic Surgeons recommends against viscosupplementation and felt the evidence to be inclusive regarding corticosteroid injections for osteoarthritis [14].
Surgical treatment for end stage osteoarthritis is limited. Total joint replacement is generally considered to be effective for short and long-term pain relief and usually achieves positive clinical and functional outcomes [15,16]. However, surgical treatments are not without risk of complications. These include systemic complications such as pulmonary embolism, but also local complications such as dislocation of hips, wound and joint infection, periprosthetic fracture, aseptic loosening, patellar maltracking, rupture of the extensor mechanism, stiffness with reduced range of motion, heterotopic ossification, metal hypersensitivity, vascular injury or bleeding, or nerve palsy. Success rates vary but as many as 1 in 5 patients undergoing total knee arthroplasty (TKA) are not satisfied with the outcome [16]. Obese patients are at increased risk for complications following TKA. Pre-operative management including weight loss, optimization of diabetes treatment, venous thromboembolism prevention, and physical therapy can help to minimize these complications [17]. Even so, some individuals will not be surgical candidates. Clearly other options are needed for effective pain relief to minimize disability and optimize function in patients who are not candidates for surgery and for whom standard analgesics have not been helpful.
Palliative therapy is specialized medical care focused on providing relief from the symptoms and stress of a serious illness. The goal is to improve quality of life and enhance physical function, but without treating or attempting to cure the underlying disease. Palliative therapy for end stage osteoarthritis is a concept that has been explored but due to a lack of effective therapies has not been very successful [18].

Neurobiology of chronic pain
Pain is the result of nerves transmitting a noxious signal, usually the result of some sort of injury, to the brain where it is perceived as pain. This is an important signal for the organism experiencing the injury to withdraw or avoid the stimulus that is producing the pain. Chronic pain results when nociceptive systems are altered so that there is no longer a direct relationship between a noxious stimulus and pain perception. These alterations are due to plasticity of the nervous system whereby peripheral nerves become sensitized, or spinal cord neurons become increasingly excitable. Projections from the spinal cord to higher centers can result in changes to descending inhibitory controls that are initiated in the midbrain and brainstem. All these changes together tend to alter the perceived response to any stimulus and thus lead to persistent pain states. This plasticity appears to be reversible and thus amenable to pharmacologic therapies [19].
Peripheral sensitization is thought to be the result of inflammation or nerve injury which alters nociceptive receptors causing increased intracellular calcium and activated intracellular protein kinase C and tyrosine kinases. These mediators phosphorylate sensory neuron-specific sodium channels and Transient Receptor Potential Vanilloid 1 (TRPV1) receptors causing a reduction in the depolarization threshold and reduced pain threshold. Nociceptive neurons themselves release chemical stimulants such as substance P (SP) and calcitonin gene-related peptide (CGRP) which amplify the local inflammatory response by interacting with local inflammatory cells and nearby blood vessels. This "neurogenic inflammation" causes vasodilation and edema, and increases local inflammation adding to peripheral sensitization [20]. Pharmacologic inhibition of this sensitization process is an attractive target for analgesia, as reducing sensitization would be expected to reduce the pain perception without eliminating the important pain defense mechanisms. Given the critical involvement of neuropeptides in the development of sensitization, the efficacy of neurotoxins was hypothesized for treatment of chronic pain.

Botulinum toxin background and human studies
There are eight serotypes of botulinum toxin. All are products of the bacterial genus Clostridium. Types A-G have been fully characterized and have varying durations of action, and enzymatic targets. They all cleave components of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins. The inability of the disrupted SNARE proteins to bring the synaptic vesicle membrane and the terminal plasma membrane of the peripheral nerve in close proximity results in an inability of the two membranes to fuse and failure of the nerve to release neurotransmitter such as acetylcholine (ACh). This produces the dramatic paralytic activity of botulinum toxin [21]. The eighth serotype, H, has been recently described, but its gene sequence has been withheld due to public safety concerns since it is considered the deadliest substance in the world [22].
Botulinum toxins A and B have been used for some time to treat painful muscle dystonias such as torticollis. It was thought that the paralytic effect of the toxin on motor units in the dystonic muscle was responsible for the pain relief that accompanied this treatment. But it was observed that pain relief preceded the muscle weakness that was expected with these treatments. This observation led to early studies of the use of intra-articular onabotulinum toxin (Type A) for end stage osteoarthritis [23]. Subsequent similar studies have been done and summarized in meta-analyses and systematic reviews. Their findings suggest that even for end stage arthritis pain, intra-articular botulinum toxin has modest beneficial effects in patients with refractory joint pain [24][25][26][27]. Studies of shoulders and knees predominated but one study treated refractory ankle osteoarthritis pain and one treated refractory pain after total knee arthroplasty. Doses used were between 100 and 200 IU onabotulinum toxin A (BOTOX), 200-500 IU abobotulinum toxin A (Dysport) and 2500 IU rimabotulinum toxin B (Myobloc). Controls in these studies were variable ranging from triamcinolone to saline to unspecified placebo. Some studies used botulinum toxin diluted with lidocaine and compared to saline with lidocaine. One small study of 75 patients compared intra-articular (IA) botulinum toxin A to injection with 2 ml sodium hyaluronate in patients with symptomatic ankle OA and found no difference in effectiveness between the two interventions [28]. Since the American

From Conventional to Innovative Approaches for Pain Treatment
Academy of Orthopedic Surgeons has stated in their evidence based guidelines for the treatment of OA of the knee that viscosupplementation cannot be recommended, this comparison may be less than appropriate [29].
Studies of IA botulinum toxin use in humans have not reported significant safety issues. Although weakness was initially a concern, it was not found in extensive safety evaluations [23,26]. Since a single injection provides pain relief for up to 6 months, fewer injections may be required as compared to corticosteroid or viscosupplementation injections and therefore, the risk of infection is minimized.

Botulinum toxin studies in preclinical models of joint pain
In an effort to better understand the mechanism of action of pain relief seen with botulinum toxin and to precisely define functional outcomes, a variety of animal studies have been done ( Table 1). Botulinum toxins have been given intraarticularly for joint pain in mice, rats and dogs. IA botulinum toxin appears to be effective for chronic arthritis pain but not acute joint pain in mice, supporting the idea that botulinum toxin reduces peripheral sensitization by inhibiting neuropeptide release in the periphery [30,31]. Only one study evaluated efficacy of rimabotulinum toxin for osteoarthritis pain in mice and found that it reduced both spontaneous and evoked pain behaviors [32]. In dogs with chronic lameness due to stifle, hip or elbow osteoarthritis IA onabotulinum toxin produced improvement in several force platform variables including vertical impulse, peak vertical force and in the Helsinki chronic pain index compared to the placebo group after 12 weeks. The secondary outcomes of subjective pain score and the need for rescue analgesics were not significantly improved in the botulinum toxin treated group compared to placebo. No major adverse events were detected [33]. A second study in dogs designed to detect adverse effects of IA botulinum toxin compared toxin injection to placebo in healthy beagle dogs. This study evaluated dynamic and static weight bearing, range of motion, joint tenderness, synovial fluid, neurologic function and electrophysiologic recordings, and histopathology of joint structures and adjacent muscles and nerves. Intra-articular botulinum toxin A did not produce significant clinical, cytological, or histopathological adverse effects in healthy dogs, but based on the electrophysiological recordings that found low compound muscle action potentials in 2 dogs in the botulinum toxin injected limb, the authors concluded that toxin may spread from the joint, but that its clinical impact is probably low [34]. In rats with inflammatory arthritis of the temporal mandibular joint (TMJ) produced by immunization with bovine serum albumin (BSA) and subsequent intra-articular challenge with BSA, injecting the joint with botulinum toxin A significantly reduced nociceptive behaviors that resulted from IA injection of low dose formalin into these inflamed joints. These authors demonstrated that the trigeminal ganglion of botulinum A treated arthritic animals released less substance P (SP) and calcitonin gene related peptide (CGRP) than saline treated arthritic animals but glutamate release was not affected. Glutamate receptors AMPA and NMDA were also unchanged in botulinum treated ganglia compared to saline treated controls. Periarticular tissues from the arthritic TMJs released increased amounts of interleukin 1-β (IL-1β) and tumor necrosis factor α (TNFα). Treatment with botulinum toxin reduced IL-1β release but had no effect on TNFα [35]. In another study of rats with adjuvant-induced arthritis induced in the tibial-tarsal joint, mechanical and thermal hyperalgesia and TRPV1 expression in the L4-5 dorsal root ganglia (DRG) were measured. DRGs were also stained for the presence of cleaved synaptosomal-associated protein of 25 kDa (SNAP-25)-the cleavage product of botulinum toxin A-and for transient receptor potential vanilloid 1 and put TRPV1 in parentheses TRPV1 and CGRP. TRPV1 expression increased significantly in the arthritic animals' DRGs and arthritic animals demonstrated mechanical and thermal hyperalgesia. Botulinum toxin A increased the paw withdrawal threshold and latency to both mechanical and thermal stimuli and reduced TRPV1 expression in a dose-dependent manner. TRPV1 transcription was likewise increased with Complete Freund's Adjuvant (CFA) induced arthritis but botulinum toxin A did not alter this increased transcription. Using immunofluorescent staining, these authors found that the increase in TRPV1 and CGRP co-expressing neurons which was the result of CFA arthritis was reduced by botulinum toxin in a dose dependent manner. Since botulinum toxin exerts its effects by cleaving SNAP-25 and thus preventing  release of neuropeptide by preventing fusion of vesicles with the terminal membrane, the presence of cleaved SNAP 25 localized with TRPV1 in the DRG was analyzed. Co-localization of cleaved SNAP-25 with TRPV1 in the botulinum toxin A group was clearly seen 5 days after botulinum toxin injection. This was not seen in the sham and CFA saline control groups. These authors speculated that botulinum toxin A may prevent TRPV1 expression on DRG neurons by inhibition of TRPV1 trafficking to the cell membrane after retrograde transport of botulinum toxin from the periphery to the DRG since the expression of the TRPV1 receptor has been shown to be dependent on exocytosis that requires interactions with proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of SNAP-25 [36]. The presence of botulinum toxin in the DRG would prevent the SNARE complex from functioning normally to move TRPV1 to the cell membrane. A botulinum toxin A based molecule-BiTox-has been synthesized that is reported to retain neuronal silencing capacity without causing paralysis. This molecule reduces plasma extravasation and inflammatory edema but is not transported to the DRG ganglia or dorsal horn and does not inhibit pain behaviors in response to formalin or capsaicin and does not inhibit formalin-induced c-Fos expression in the dorsal horn. It was found to strongly reduce A-nociceptor mediated secondary mechanical hyperalgesia due to CFA joint inflammation or capsaicin injection and decreased hypersensitivity from nerve injury. The authors concluded that this botulinum toxin based molecule could reduce local release of neuromodulators from C fibers without impairing C nociceptive signaling function [37].

Vanilloids as analgesics 4.1 Vanilloids and their receptors background
Vanilloids such as capsaicin (the active ingredient in hot chili peppers) and resiniferatoxin (a product of the plant Euphorbia resinifera) were first notable for their ability to produce burning pain when administered topically. Later, both molecules were found to have analgesic potential and subsequent work identified the non-selective cation channel to which these compounds bind. This receptor was found to be located on the dorsal root and trigeminal ganglia of various species. Subsequent work identified the channel, allowed cloning and cDNA characterization, and revealed that the channel could be activated not only by vanilloids but also by heat suggesting a role in thermosensation. The receptor was named transient receptor potential vanilloid 1 (TRPV1) and other ligands were identified making it a transducer of many types of noxious stimuli [38].
Because of the variety of ligands, TRPV1 was considered a likely target for analgesia. Agonists such as vanilloids were noted to cause desensitization of these channels and in rodents as well as humans, pain behaviors could be alleviated with vanilloid treatment. TRPV1 knockout mice demonstrated reduced thermal hypersensitivity with inflammation. TRPV1 antagonists were shown to reverse pain behavior in rodents with a wide variety of painful conditions including inflammation, osteoarthritis and cancer. Both agonists and antagonists have been considered as analgesic therapies. Although systemic administration of vanilloids demonstrated analgesic efficacy in preclinical pain models, because of the undesirable systemic side effects of these compounds, most therapeutic trials have focused on local or topical administration of these compounds. Undesirable effects include hypotension, respiratory compromise and other negative effects on reflex pathways. Less pungent analogs were found to be less efficacious with respect to analgesia [38].

Vanilloids as analgesics-Human and pre-clinical studies
In humans, current treatment of joint pain with vanilloids is limited to topical therapies. Pain relieving creams such as Zostrix ® and patches such as Salonpas Hot Capsicum Patch ® are available over the counter and contain capsaicin as the active ingredient. Multiple clinical trials have found modest benefit for osteoarthritis from low dose topical capsaicin [39][40][41]. More recently, a high dose 8% capsaicin patch has been approved for the treatment of post-herpetic neuralgia. According to the package insert, application of the patch requires careful adherence to application instructions by the health care professional and local anesthesia for the patient prior to application and systemic analgesics as needed in the post-application period. Interestingly, results from clinical trials of this drug for treatment of painful HIV neuropathy did not show clinical benefit [42,43].
Resiniferatoxin (RTX) is an ultra-potent capsaicin (CAP) analogue [44], that is several thousand-fold more potent than CAP [45]. RTX in low concentrations produces a slow and sustained depolarization of membrane potential, preventing the generation of action potentials but causing less toxicity. A single IA injection in rats has been found to reduce hyperalgesia due to carrageenan induced joint pain [46].
RTX has been studied in clinical trials for other painful conditions. When given intravesicularly for interstitial cystitis and painful bladder syndrome, it did not improved overall symptoms of pain, urgency, frequency or nocturia [47,48]. Adlea™ (4975) is another CAP analogue under development for the treatment of post-operative musculoskeletal pain, osteoarthritis and tendinopathy. Phase II trials of intra-articular injection of this compound were encouraging but no further clinical trials appear to have been performed [38]. Zucapsaicin (Civamide) is the cis-isomer of capsaicin, and functions as a TRPV1 blocker. Phase III trials have been done with topical civamide for OA knee pain [49]. This topical therapy is not absorbed systemically, is well tolerated, and produced significant improvement in Western Ontario and McMaster Universities Arthritis Index (WOMAC) physical function score, pain score and subject global evaluation. Improvement was maintained for a year. This drug has not yet been approved by the US FDA.

TRPV1 antagonists as analgesics
Several TRPV1 antagonists have also been identified that act as analgesics [50]. Some are more selective than others, complete nonselectivity producing inhibition of all modes of TRPV1 activation (protons, heat and capsaicin). More selectivity  appears to improve the side effect profile. Centrally active TRPV1 antagonists appear to provide greater analgesia when given systemically or intrathecally in preclinical models of OA pain. Most preclinical studies of OA have been done in the monosodium iodoacetate (MIA) model in the rat ( Table 2). Rats with MIA-induced arthritis pain demonstrate reduced weightbearing of the affected limb, pain with movement of the joint and hypersensitivity of uninjured tissues (secondary allodynia of the hind paw). These pain behaviors are thought to be due to both central and peripheral sensitization. TRPV1 antagonists appeared to be analgesic during both the acute inflammatory phase of MIA pain and later during the chronic phase [50][51][52][53]. Analgesia appears to improve with repeated dosing and side effects such as hyperthermia abate with some of the investigational TRPV1 antagonists. The potency of hyperthermia induction seems to relate most closely to the blockade of proton-induced TRPV1 activation [50]. Only a few TRPV1 antagonists have been used in clinical trials. ADZ1386 given orally in two different doses did not reduce OA pain more than placebo. A study in dogs with hip OA using oral ABT116 did not improve the total pain score, pain severity or pain interference score, but did reduce rescue medication use, increased night time activity levels and briefly produced an acute hyperthermic effect. NEO 6860, which is specific for blocking capsaicin activation of the target, with little or no effect against pH or heat activation, underwent a first-in-human phase I trial of the safety and efficacy of the drug in healthy human subjects [54]. The dose ranging study included 64 subjects and measured pharmacodynamics with a intradermal capsaicin test as well as pharmacokinetics. The drug was rapidly absorbed with a half-life of between 4 and 8 hours. Side effects included headache, paresthesia, nausea, and dizziness. Study participants were monitored specifically for increase in temperature and heat pain threshold/tolerance, but these were not noted. At all doses, most subjects reported a rapid onset, transient sensation of "feeling hot". The authors concluded that this compound had potential for development for treating OA-associated pain and future clinical studies were planned but have not yet been initiated.

Conotoxin
Ziconotide (ω-conopeptide MVIIA) is a synthetic compound of the neurotoxin ω-conopeptide derived from the Conus Magus fish hunting marine snail found in the Pacific Ocean. It selectively binds to the N-type voltage-gated calcium channels found in the laminae of the spinal cord's dorsal horn and blocks these channels. This blockade prevents calcium influx and halts neurotransmission thereby preventing nociceptive signaling. Pain transmission messages are prevented from arriving at the brain. It is FDA approved for intrathecal use for severe chronic pain in individuals who are intolerant of or refractory to other treatments including intrathecal (IT) morphine, but has demonstrated some serious side effects such as suicidal ideation and psychosis [55].
In a study of transgenic mice bred to express a membrane-tethered form of the conotoxin ω-conopeptide MVIIA under control of a nociceptor-specific gene, who were subjected to unilateral induction of joint inflammation with the antigen-and collagen-induced arthritis (ACIA) model, pain was effectively suppressed, but joint inflammation became persistent and more destructive. The authors concluded that blockade of Ca V 2.2-mediated calcium influx and nociceptive signaling by this toxin impaired recovery from induced inflammatory arthritis. They concluded that this blockade could lead to potentially deleterious and devastating effects if used during inflammation [55].

Tetrodotoxin
Another neurotoxin studied as a potential analgesic is tetrodotoxin (TTX). Voltage-gated sodium channels (VGSCs) are critical for neuronal function and dysfunctional VGSCs have been implicated in several pain states. There are nine isoforms of the sodium channel alpha-subunit (Nav1.1-1.9 in mammals). Only Nav1.1-1.4 and Nav1.6-1.7 subtypes (TTX-sensitive channels) can be blocked by nanomolar concentrations of tetrodotoxin. Micromolar concentrations are required to block Nav1.5 and Nav1.8-1.9 subtypes (TTX-resistant channels) [56]. Although it appears to have little effect on acute pain further studies are needed. Analgesic efficacy in preclinical inflammatory pain models demonstrated promising effects of systemic administration for mechanical hyperalgesia and the neurogenic inflammatory response to injury. There are contradictory results for TTX efficacy for neuropathic pain. Effectiveness in preclinical models of neuropathic pain varied depending on dose, route of application, and appeared more effective in acute neural injury than in chronic neuropathic pain. In one clinical trial of tetrodotoxin for chemotherapy-induced neuropathic pain, injected TTX did not have a significant effect on pain [57]. There have been no specific studies evaluating tetrodotoxin for the treatment of chronic joint pain.

Anti-nerve growth factor
Thought not technically neurotoxins, several monoclonal antibodies have been developed against nerve growth factor (NGF) specifically for the treatment of chronic pain, and specifically for pain from OA. Tanezumab was the first of these to be developed. Three other companies have now created similar antibodies. Tanezumab was in phase III studies when the US FDA placed a hold on further clinical trials after an increase in joint destruction was observed in patients who had been given this drug. After that, preclinical studies suggested that this class of drugs could damage the autonomic nervous system, which delayed further research [58]. Since the hold was released in 2015, phase III clinical trials are being repeated. Results from those that have been published show that these biologic therapies appear to be effective with acceptable side effect profiles [59][60][61]. These therapies are administered parenterally, and therefore are systemically active. These antibodies have significant potential to improve analgesia for chronic arthritis pain. Alternative routes of administration such as IA will be of interest.

Conclusions
Chronic joint pain is a significant public health problem that will only increase along with the aging population. In the absence of disease modifying treatments for OA, the need for better pain therapies will continue to increase. Neurotoxins can be helpful as adjunct treatments for pain, particularly in cases where peripheral sensitization has lowered pain thresholds and increased pain perception. Advances in understanding of the pathophysiologic mechanisms of nociception and sensitization and elucidation of the specific functions of the various neurotoxins will allow more advanced development of toxins that may avoid potential side effects and more specifically reduce pain perception.