Botulinum Toxin: An Update on Pharmacology and Newer Products in Development

Since its introduction as a treatment for strabismus, botulinum toxin (BoNT) has had a phenomenal journey and is now recommended as first-line treatment for focal dystonia, despite short-term clinical benefits and the risks of adverse effects. To cater for the high demand across various medical specialties, at least six US Food and Drug Administration (FDA)-approved formulations of BoNT are currently available for diverse labelled indications. The toxo-pharmacological properties of these formulations are not uniform and thus should not be used interchangeably. Synthetic BoNTs and BoNTs from non-clostridial sources are not far from clinical use. Moreover, the study of mutations in naturally occurring toxins has led to modulation in the toxo-pharmacokinetic properties of BoNTs, including the duration and potency. We present an overview of the toxo-pharmacology of conventional and novel BoNT preparations, including those awaiting imminent translation from the laboratory to the clinic.     

New approaches to discovering drugs that treat dystonia

ABSTRACT

Introduction: Dystonia consists of involuntary movements, abnormal posturing, and pain. In adults, dystonia presents in a particular region of the body and causes significant disability due to pain as well as impairment in activities of daily living and employment. The current gold standard treatment, botulinum toxin (BoNT), has limitations – painful, frequent injections due to ‘wearing off’ of treatment effect; expense; and expected side effects like swallowing difficulty and weakness. There is a clear therapeutic gap in our current treatment options for dystonia and also a clear need for an effective novel treatment. Testing any novel treatment is complicated because most adults with focal dystonia are treated with BoNT.

Areas covered: This review focuses on establishing the need for novel therapeutics. It also suggests potential leads from preclinical studies; and, discusses the issue of clinical trial readiness in the dystonia field.

Expert opinion: Identifying a novel therapeutic intervention for dystonia patients faces two major challenges. The first is acknowledging the therapeutic gap that currently exists. Second, shifting some of our research aims in dystonia to clinical trial readiness is imperative if we are to be ready to test novel therapeutic agents.     

Botulinum toxin therapy for pain and inflammatory disorders: mechanisms and therapeutic effects

Botulinum toxin (BTX) injections are a well-recognised therapeutic modality for the treatment of regional involuntary muscle disorders and recently BTX has been used for treatment of pain and inflammatory disorders. The primary purpose of this review is to discuss the mechanism of action of therapeutic BTX in light of both the traditional understanding of BTX pharmacological effects as well as new observations. The review will deal with clinical observations and relevant animal experimentation. The data and hypotheses presented are not only relevant to botulinum toxin technology but will certainly prove important in the basic mechanisms of some of the diseases where botulinum toxin has been successfully applied. BTX used clinically comprises botulinum neurotoxin (BoNT) complexed with non-toxic proteins. The non-toxic components of the BTX complexes stabilise the labile BoNT during purification and formulation as a therapeutic. The complex proteins may also have unrecognised clinical significance such as slowing diffusion in tissues or imparting stability. The mechanisms of BTX formulations acting on SNARE proteins are briefly reviewed providing a basis for BTX clinical applications. The potential for design of improved botulinum toxins and formulations is addressed.     

Clinical Uses of Botulinum Neurotoxins: Current Indications,Limitations and Future Developments

Botulinum neurotoxins (BoNTs) cause flaccid paralysis by interfering with vesicle fusion and neurotransmitter release in the neuronal cells. BoNTs are the most widely used therapeutic proteins. BoNT/A was approved by the U.S. FDA to treat strabismus, blepharospam, and hemificial spasm as early as 1989 and then for treatment of cervical dystonia, glabellar facial lines, axillary hyperhidrosis, chronic migraine and for cosmetic use. Due to its high efficacy, longevity of action and satisfactory safety profile, it has been used empirically in a variety of ophthalmological, gastrointestinal, urological, orthopedic, dermatological, secretory, and painful disorders. Currently available BoNT therapies are limited to neuronal indications with the requirement of periodic injections resulting in immune-resistance for some indications. Recent understanding of the structure-function relationship of BoNTs prompted the engineering of novel BoNTs to extend therapeutic interventions in non-neuronal systems and to overcome the immune-resistance issue. Much research still needs to be done to improve and extend the medical uses of BoNTs.     

Development of future indications for BOTOX

Since the late 1970s, local injections of BoNT have provided clinical benefit for patients with inappropriately contracting muscles with or without pain or sensory disturbance. Marketing authorization for some BoNTs, depending on country, include core indications of dystonia (blepharospasm and cervical dystonia), large muscle spastic disorders (not yet approved in the United States, e.g., adult post-stroke spasticity and equinus foot deformity), hyperhidrosis and aesthetic. Subsequent development has extended to selected conditions characterized by recurrent or chronic pain (migraine headache), and urologic indications (neurogenic/idiopathic overactive bladder; prostate hyperplasia), with multiple additional opportunities available. Portfolio management requires a careful individual opportunity assessment of scientific and technical aspects (basic science foundation, potential to treat unmet medical need, product-specific risk in specific populations, therapeutic margin/safety profile, and probability of successful registration pathway). This article describes ongoing development targets for BOTOX^®.     

Pain Relief in Cervical Dystonia with Botulinum Toxin Treatment

Dystonia is a neurological disorder characterized by intermittent or sustained muscle contractions that cause abnormal, usually repetitive, movements and postures. Dystonic movements can be tremulous and twisting and often follow a pattern. They are frequently associated with overflow muscle activation and may be triggered or worsened by voluntary action. Most voluntary muscles can be affected and, in the case of the neck muscles, the condition is referred to as cervical dystonia (CD), the most common form of dystonia. The high incidence of pain distinguishes CD from other focal dystonias and contributes significantly to patient disability and low quality of life. Different degrees of pain in the cervical region are reported by more than 60% of patients, and pain intensity is directly related to disease severity. Botulinum toxin (BoNT) is currently considered the treatment of choice for CD and can lead to an improvement in pain and dystonic symptoms in up to 90% of patients. The results for BoNT/A and BoNT/B are similar. The complex relationship between pain and dystonia has resulted in a large number of studies and more comprehensive assessments of dystonic patients. When planning the application of BoNT, pain should be a key factor in the choice of muscles and doses. In conclusion, BoNT is highly effective in controlling pain, and its analgesic effect is sustained for a long time in most CD patients.     

Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM

Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.     

Disease-oriented approach to botulinum toxin use

Botulinum toxin (BoNT) has been used for over a quarter of century for the treatment of well over 100 different indications. Many of the symptoms for which BoNT has been found to be effective occur in a variety of neurological disorders. One neurodegenerative disorder in which BoNT has been used extensively to treat various symptoms is Parkinson's disease (PD). This review will highlight the following therapeutic applications of BoNT in conditions associated with PD: limb dystonia, blepharospasm and lid apraxia, bruxism, cervical dystonia (anterocollis), camptocormia, hand and jaw tremor, rigidity (painful shoulder), freezing of gait, sialorrhea, dysphagia (achalasia), seborrhea, hyperhidrosis, overactive bladder, and constipation.     

Botulinum Toxin for Neuropathic Pain: A Review of the Literature

Botulinum neurotoxin (BoNT), derived from Clostridium botulinum, has been used therapeutically for focal dystonia, spasticity, and chronic migraine. Its spectrum as a potential treatment for neuropathic pain has grown. Recent opinions on the mechanism behind the antinociceptive effects of BoNT suggest that it inhibits the release of peripheral neurotransmitters and inflammatory mediators from sensory nerves. There is some evidence showing the axonal transport of BoNT, but it remains controversial. The aim of this review is to summarize the experimental and clinical evidence of the antinociceptive effects, mechanisms, and therapeutic applications of BoNT for neuropathic pain conditions, including postherpetic neuralgia, complex regional pain syndrome, and trigeminal neuralgia. The PubMed and OvidSP databases were searched from 1966 to May 2015. We assessed levels of evidence according to the American Academy of Neurology guidelines. Recent studies have suggested that BoNT injection is an effective treatment for postherpetic neuralgia and is likely efficient for trigeminal neuralgia and post-traumatic neuralgia. BoNT could also be effective as a treatment for diabetic neuropathy. It has not been proven to be an effective treatment for occipital neuralgia or complex regional pain syndrome.     

Treatment of Depression with Botulinum Toxin

Injection of botulinum toxin (BoNT) into the glabellar region of the face is a novel therapeutic approach in the treatment of depression. This treatment method has several advantages, including few side effects and a long-lasting, depot-like effect. Here we review the clinical and experimental evidence for the antidepressant effect of BoNT injections as well as the theoretical background and possible mechanisms of action. Moreover, we provide practical instructions for the safe and effective application of BoNT in the treatment of depression. Finally, we describe the current status of the clinical development of BoNT as an antidepressant and give an outlook on its potential future role in the management of mental disorders.     

Botulinum Toxin as a Pain Killer: Players and Actions in Antinociception

Botulinum neurotoxins (BoNTs) have been widely used to treat a variety of clinical ailments associated with pain. The inhibitory action of BoNTs on synaptic vesicle fusion blocks the releases of various pain-modulating neurotransmitters, including glutamate, substance P (SP), and calcitonin gene-related peptide (CGRP), as well as the addition of pain-sensing transmembrane receptors such as transient receptor potential (TRP) to neuronal plasma membrane. In addition, growing evidence suggests that the analgesic and anti-inflammatory effects of BoNTs are mediated through various molecular pathways. Recent studies have revealed that the detailed structural bases of BoNTs interact with their cellular receptors and SNAREs. In this review, we discuss the molecular and cellular mechanisms related to the efficacy of BoNTs in alleviating human pain and insights on engineering the toxins to extend therapeutic interventions related to nociception.     

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.     

Recommended Mass Spectrometry-Based Strategies to Identify Botulinum Neurotoxin-Containing Samples

Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between 0.1 μg to 70 μg, so it is important to enable detection of small amounts of these toxins. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric‑based endopeptidase method to detect, differentiate, and quantify BoNT immunoaffinity purified from complex matrices. In this work, we describe the application of Endopep-MS for the analysis of thirteen blinded samples supplied as part of the EQuATox proficiency test. This method successfully identified the presence or absence of BoNT in all thirteen samples and was able to successfully differentiate the serotype of BoNT present in the samples, which included matrices such as buffer, milk, meat extract, and serum. Furthermore, the method yielded quantitative results which had z-scores in the range of −3 to +3 for quantification of BoNT/A containing samples. These results indicate that Endopep-MS is an excellent technique for detection, differentiation, and quantification of BoNT in complex matrices.     

Practical Application of Novel Test Methods to Evaluate the Potency of Botulinum Toxin: A Comparison Analysis among Widely Used Products in Korea

The safe and effective dosing of botulinum neurotoxins (BoNTs) requires accurate and reliable methods to measure their potency. Several novel methods have been introduced over the past decade; however, only few studies have compared the potency of BoNT products with that of the LD50 and other alternative assays. Therefore, the objective of this study was to comparatively evaluate widely used BoNT products using various test methods. Four types of BoNTs (prabotulinumtoxin A, onabotulinumtoxin A, neubotulinumtoxin A, and letibotulinumtoxin A) were used in this study. The estimated potency was assessed using the LD50 assay, and the total BoNT type A protein levels were measured using the enzyme-linked immunosorbent assay (ELISA). The in vitro efficacy of the BoNTs was determined using fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) assays. The results showed differences in the total amount of BoNT protein and the cleavage activity of SNAP-25 within all types of BoNTs. The SPR study seemed to be useful for evaluating the potency by specifically measuring intact 19S neurotoxin, and these results provide new insights for assessing different BoNT products.     

Management of bladder, prostatic and pelvic floor disorders with botulinum neurotoxin

Since its introduction in the late 1970s for the treatment of strabismus and blepharospasm, botulinum toxin (BoNT) has been increasingly used in the interventional treatment of several other disorders characterized by excessive or inappropriate muscle contractions. The use of this pluripotential agent has extended to a plethora of conditions including: focal dystonia; spasticity; inappropriate contraction in most sphincters of the body such as those associated with spasmodic dysphonia, esophageal achalasia, chronic anal fissure, and vaginismus; eye movement disorders; other hyperkinetic disorders including tics and tremors; autonomic disorders such as hyperhidrosis; genitourinary disorders such as overactive and neurogenic bladder, non-bacterial prostatitis and benign prostatic hyperplasia; and aesthetically undesirable hyperfunctional facial lines. In addition, BoNT is being investigated for the control of the pain, and for the management of tension or migraine headaches and myofascial pain syndrome. BoNT injections have several advantages over drugs and surgical therapies in the management of intractable or chronic disease. Systemic pharmacologic effects are rare; permanent destruction of tissue does not occur. Graded degrees of relaxation may be achieved by varying the dose injected; most adverse effects are transient. Finally, patient acceptance is high. In this paper, clinical experience over the last years with BoNT in urological impaired patients will be illustrated. Moreover, this paper presents current data on the use of BoNT to treat pelvic floor disorders.     

The therapeutic use of botulinum toxin

Since Alan Scott’s research, botulinum toxin (BoNT) has been used in several diseases or conditions characterised by muscular overactivity. BoNT acts on either neuromuscular or autonomic cholinergic junctions. Seven different serotypes are known, with antigenic specificity and different therapeutic profiles. BoNT is made up of a heavy chain, involved in binding and membrane translocation, and a light chain, involved in blocking neuroexcytosis. Each serotype shares a specific acceptor on the presynaptic membrane of a cholinergic junction. The available BoNT preparations differ in toxicity, purity and stability. Injection of the neurotoxin produces several modifications at a neuromuscular junction. Axonal sprouting, muscular fibre atrophy, and new end-plates are the most evident histological events after BoNT treatment. They appear to be reversible in untreated muscles. Diffusion can occur at first by haematogeneous or local BoNT spread. Several factors, such as dose, volume, site of injection, muscle size, and muscular fascia, can influence the amount of diffusion and possible side-effects. After prolonged BoNT treatment patients can become unresponsive. Antibodies directed against BoNT have been observed with ELISA or mouse bioassay. Different serotypes have been used to treat non-responder patients. Novel toxins with lower immunogenicity and prolonged clinical efficacy are required for more effective treatment.     

Alpha-Latrotoxin Rescues SNAP-25 from BoNT/A-Mediated Proteolysis in Embryonic Stem Cell-Derived Neurons

The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K(+) had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca(2+) influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery.     

Botulinum toxin for the treatment of cervical dystonia

Cervical dystonia (CD) manifests clinically through involuntary spasms of neck muscles, producing abnormal head and neck movements and postures, which is often associated with pain. CD is the most common form of focal dystonia presenting to movement disorders clinics. Chemodenervation with botulinum toxin (BTX) has become the first-line treatment for CD, producing satisfactory relief of symptoms in > 80% of cases. Unresolved issues that may impact on the overall results include the method of selection for injection sites (clinical vs. electromyography), dosing, dilution and the role and relative efficacy of the different BTX serotypes. A guiding therapeutic principle of BTX injections is to achieve optimal results with the lowest possible dosage and frequency of administration. This strategy is critical in order to keep the risk of immunoresistance at a minimum. Development of antibodies that block the effects of BTX, usually associated with frequent injections of high doses, is the main reason for secondary unresponsiveness to this treatment. Although the mechanism of denervation at the neuromuscular junction by BTX is relatively well understood, the role of changes in muscle spindles and myopathic pain mechanisms, as well as secondary changes at the level of the basal ganglia, thalamus and cortex and their role in response to BTX, all need further exploration.     

Botulinum Toxin Type A for the Treatment of Neuropathic Pain in Neuro-Rehabilitation

Pain is a natural protective mechanism and has a warning function signaling imminent or actual tissue damage. Neuropathic pain (NP) results from a dysfunction and derangement in the transmission and signal processing along the nervous system and it is a recognized disease in itself. The prevalence of NP is estimated to be between 6.9% and 10% in the general population. This condition can complicate the recovery from stroke, multiple sclerosis, spinal cord lesions, and several neuropathies promoting persistent disability and poor quality of life. Subjects suffering from NP describe it as burning, itching, lancing, and numbness, but hyperalgesia and allodynia represent the most bothersome symptoms. The management of NP is a clinical challenge and several non-pharmacological and pharmacological interventions have been proposed with variable benefits. Botulinum toxin (BTX) as an adjunct to other interventions can be a useful therapeutic tool for the treatment of disabled people. Although BTX-A is predominantly used to reduce spasticity in a neuro-rehabilitation setting, it has been used in several painful conditions including disorders characterized by NP. The underlying pharmacological mechanisms that operate in reducing pain are still unclear and include blocking nociceptor transduction, the reduction of neurogenic inflammation by inhibiting neural substances and neurotransmitters, and the prevention of peripheral and central sensitization. Some neurological disorders requiring rehabilitative intervention can show neuropathic pain resistant to common analgesic treatment. This paper addresses the effect of BTX-A in treating NP that complicates frequent disorders of the central and peripheral nervous system such as spinal cord injury, post-stroke shoulder pain, and painful diabetic neuropathy, which are commonly managed in a rehabilitation setting. Furthermore, BTX-A has an effect in relief pain that may characterize less common neurological disorders including post-traumatic neuralgia, phantom limb, and complex regional pain syndrome with focal dystonia. The use of BTX-A could represent a novel therapeutic strategy in caring for neuropathic pain whenever common pharmacological tools have been ineffective. However, large and well-designed clinical trials are needed to recommend BTX-A use in the relief of neuropathic pain.