Toxin Structure

Clostridium botulinum bacteria are gram-positive anaerobic rods that produce a family of 7 serologically distinguishable exotoxins: A, B, C1, D, E, F, and G. Only botulinum toxins (BoNTs) type A (BoNT-A) and type B (BoNT-B) are available for clinical use. All of the BoNTs produce temporary presynaptic blockade of cholinergic nerve terminals by blocking the release of acetylcholine (ACh) at neuromuscular junctions (NMJ) and neuroglandular junctions (NGJ).

The BoNTs are synthesized as a single 150-kDa chain. For bioactivity this single chain must be “nicked” or cleaved into a bichain molecule composed of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC) linked by a disulfide bond. Only some species of C botulinum contain the proteases required to nick/activate the toxin. Nonproteolytic bacteria such as those producing serotype E require trypsin to activate the toxin. The percentage of toxin B that is recovered nicked from culture is lower than that of BoNT A, which is 95% nicked. This factor is one of several which may account for the observed potency and potential immunogenic differences between and within the serotypes of BoNT.

The bichain BoNTs are folded into a complex 3-dimensional (3-D) globular shape. The shape of the individual toxins influences acceptor binding at the NMJ and proteins involved in ACh release. The size of the total complex of the different commercially available products varies from 500 to 900 kDa because of the presence of accessory molecules, including hemagglutinin (HA) and nonhemagglutinin (NH) proteins, associated with the toxin. The HA and NH proteins act to stabilize and shield the toxin. Individual BoNT products vary significantly in the manufacturing process, the strain of C botulinum toxin used for culture, and the amount of accessory protein. These differences are specified in product information provided by each manufacturer. For internalization and bioactivity, BoNTs must remain in the linked bichain form (HC-LC linked). If the LC-HC bond is cleaved before injection, the LC cannot cross the presynaptic membrane and the toxin is rendered inactive.

Bioactivity and Pharmacology

The intracellular production, transport, and release of ACh vesicles is a complex process involving a suite of SNARE proteins (Soluble N -ethylmaleimide-sensitive factor Attachment Protein Receptors). Normally, when an action potential reaches the NMJ or NGJ it induces synaptic vesicles to dock/fuse with the presynaptic membrane and release their contents of ACh into the synaptic cleft. BoNTs do not affect the synthesis, production, or intracellular packaging or transport of ACh in cholinergic nerve terminals. Rather, the toxins exert their effect by blocking synaptic vesicles from docking or binding at the NMJ, thereby preventing the release of ACh. The process of BoNT acceptor-mediated binding and vesicle blocking is a multistep process and includes:

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  Step 1: Binding. The C-terminus of the BoNT HC binds avidly to serotype=specific binding sites on presynaptic cholinergic nerve terminals called active zones. The N-terminus of the HC contributes to internalization of the toxin (see later discussion). Evidence suggests this process is dependent on Ca ²⁺ binding and the involvement of synaptotagmin, one of the SNARE proteins.

  
  
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  Step 2: Internalization of BoNT. BoNT is endocytosed forming an enclosed endosome formed by the lipid bilayer of the cell membrane.

  
  
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  Step 3: Translocation of the LC. For activity the LC must exit the endosome moving into the cytosol to reach its SNARE target. Once endocytosed the HC is cleaved from the LC by a mechanism that has not yet been discovered. The nitrogen or N-terminus of the HC acts as a transmembrane chaperone for the LC by creating a conducting channel or membrane pore in the endosome. The conducting channel created by the HC allows the LC to translocate the lipid bilayer of the endosome and move into the cytosol. Because of its size and 3-D globular shape, the LC must unfold before exiting through this membrane pore. This unfolding process is facilitated by a pH differential existing between the cytosol and endosome. Typically, a lower pH increases the helical structure of proteins and a higher pH facilitates unfolding. Unlike most proteins, a lower pH facilitates unfolding of the BoNT LC. The pH of 5 inside the endosome is lower than the neutral pH of the surrounding cytosol (pH 7). This lower pH allows the LC, contained in the endosome, to unfold to exit into the cytosol. Once free in the cytosol, with its higher pH, the LC refolds back into its original 3-D globular shape/structure.

Each serotype of botulinum toxin works by enzymatic cleavage of one or more of the SNARE proteins. The 3-D shape/conformation of each LC serotype is specific for corresponding sites/proteins in the SNARE complex ( Fig. 1 ). The LCs of each of the 7 botulinum serotypes cleaves a distinct peptide bond on one or more of the SNARE proteins. No 2 serotypes act at the same peptide bond. Botulinum toxins A, C, and E cleave the 25-kDa synaptosomal-associated protein SNAP-25; types B, D, F, and G cleave a synaptobrevin vesicle-associated membrane protein (VAMP).

Exocytosis of BoNT: Free vesicles ( A ) undergo docking at active zones ( B and C ), and fuse with the plasma membrane after a Ca ²⁺ influx ( D1 ). During this sequence of events, the clostridial neurotoxin targets (the 3 SNAREs VAMP, SNAP-25, and syntaxin) assemble in complexes organized in a fusogenic ring ( D2 ). D2 represents a cross section of D1 at the junction of the vesicle and plasma membranes. An open configuration is adopted by VAMP, SNAP-25, or syntaxin only during step B. This would determine the “physiologic window” during which the clostridial neurotoxins can cleave their targets. Cleaved SNAREs can form complexes (here denoted by faded shading) and incorporation of these into the fusogenic ring ( D1, D2 ) is sufficient to prevent the fusion process.

From Humeau Y, Doussau F. How botulinum and tetanus neurotoxins block neurotransmitter release. Biochimie 2000;82(5):437; with permission.

In recent years, evidence has shown that all synaptic neurotransmitter release, including nociceptive neuropeptides, is regulated by this same vesicular release mechanism involving SNARE proteins forming a membrane pore to allow release of a vesicle contents ( Fig. 2 ). This mechanism provides a theoretical basis for the observed effects of BoNTs on pain. The antinociceptive effects of the BoNTs may be produced by blocking SNARE proteins. This block may reduce the release of nociceptive neuropeptides and neurotransmitters involved in pain signaling, chronic inflammatory pain response, and central sensitization of pain. The neuropeptides and neurotransmitters involved in nociception include substance P, glutamate, calcitonin gene-related peptide, and others.

Exocytosis, SNARE proteins and vesicle docking.

Data from Martin TF. Stages of regulate exocytosis. Trends Cell Biol 1997;7:271–6; with permission.

Clinical Effects

Once the SNARE proteins are cleaved, the ACh-containing vesicles cannot dock with or be released from presynaptic membrane, thereby blocking muscle activation or gland stimulation. A corresponding decrease in muscle activity or glandular secretion occurs following blocking. Initial effects are generally noted within 48 to 72 hours with peak effect of the toxin at 4 weeks. The effects of BoNTs are temporary and begin wearing off as the process of reinnervation occurs, reestablishing a functional NMJ/NGJ. The clinical effects gradually begin wearing off as the NMJ/NGJ is reestablished. Effects typically last 3 months but may last much longer in some patients and/or conditions. Retreatment is determined by recurrence of clinical symptoms or problems, but is not recommended before 12 weeks.

Immunogenicity

The BoNTs, like all foreign proteins, can stimulate an immune response and the formation of neutralizing antibodies. The incidence of neutralizing antibodies has become less frequent in recent decades with changes in injection patterns and toxins. The problem of secondary nonresponse (defined as an absent or markedly decreased response in a patient with prior good clinical response) may be caused by issues other than antibody formation. These issues include contractures, change in pattern, and evolution of the disorder. Many patients have been treated successfully for well over a decade with no evidence of secondary nonresponse or antibody formation. When true nonresponse is considered likely, a clinical test for nonresponse rather than antibody titers is most commonly performed. The most commonly performed clinical test is the Frontalis test, which involves injecting a small quantity of BoNT in the frontalis muscle and observing the clinical response/nonresponse (ability to elevate forehead). An alternative is measurement of the compound motor action potential (CMAP) of the abductor digiti minimi pre and post toxin injection. To minimize immune system stimulation repeat BoNT treatment is performed no more frequently than at 12-week intervals. Booster dosing or dosing at intervals shorter than 12 weeks should be avoided. Although there is no specific evidence to support it, most experienced clinicians also recommend avoiding immunizations within 2 weeks of BoNT injections.

Dosing

The BoNTs are biologic agents, and dosing must be customized for the patient’s clinical problem, severity of the tone issues, size of the muscle, and the treatment goals. This customization involves both the art and science of patient evaluation, target identification, and dose selection. Using the minimal effective dose is recommended to avoid immune stimulation and adverse events such as local or regional weakness. The BoNT’s are dosed in bioactive units rather than in weight or volume of toxin. These units of potency are specific for each individual toxin manufacturer; this is true both within and between the different serotypes of toxins that are clinically available. There are no accepted conversion tables when comparing individual products.

Adverse Events

There have been reports of hypersensitivity and anaphylactic reactions to BoNT. History of these events is a contraindication to additional treatment with BoNTs. Reported adverse events include dry mouth and dysphagia, primarily seen in patients with cervical dystonia. Excess weakness in the injected muscle is generally a dosing issue. Weakness may be seen in muscles adjacent to the target or, less commonly, more distant from the site of injection. There have been some case reports suggesting spread of the toxin to sites distant from that injected. Symptoms have included asthenia, diplopia, blurred vision, ptosis, urinal incontinence dysphagia, exerostomia, and breathing problems (see the Food and Drug Administration [FDA] label update).

2010 Toxin Update

Worldwide, the number of botulinum toxin preparations has expanded to include several A serotypes and one B serotype ( Table 1 ). The inherent differences between and even within serotypes requires clinicians have a thorough knowledge of toxin biopharmacology. The differences in potency and dosing between and within serotypes are likely due to manufacturing differences, intracellular targets, and the accessory or accompanying molecules/proteins. Because the BoNTs are biologicals, not drugs, the product from each manufacturer is unique. From a clinical standpoint this mandates dosing that is specific or unique to the individual products. Despite being used in clinical practice, there are no accepted or recommended conversion factors when moving from one toxin product to another. When switching between or within serotypes it is recommended that clinicians return to the recommended starting dose for the new product.

FDA Updates

In 2009, to reduce the potential for confusion between the different toxin products, the FDA issued unique generic names for each of the 3 botulinum toxin products available in the United States (see Table 1 ). Clinicians and researchers should become familiar with these generic names.

In 2009 the FDA also issued a boxed warning for all BoNT products used in the United States. This warning includes information on possible adverse events and side effects of toxin therapy, including spread of the toxin with distant effects, weakness, swallowing problems, and death. The deaths reported in association with BoNT therapy were in compromised or medically fragile children with cerebral palsy and preexisting swallowing problems. In its investigation, the FDA did not determine a causal link between toxin therapy and the patient deaths. The FDA has mandated that a Risk Evaluation and Mitigation Strategy (REMS) be performed at each treatment session. It is mandated that physicians provide information about the risk of treatment to all patients at each treatment session.

Clinical Applications of the Botulinum Toxins

From the initial approval of botulinum toxin A (oculinum toxin) by the FDA in 1989 for ocular muscle disorders (strabismus and blepharospasm), there has been a seemingly ever-expanding list of approved indications, clinically accepted and proposed uses for the neurotoxins.

A recent Medline search for “Botulinum toxin injection” revealed over 4000 citations in peer-reviewed publications since the mid 1980s. Most physicians are familiar with the approved indications for the botulinum toxins (see Table 1 ). Many clinicians are also familiar with some of the off-label but accepted clinical uses of the botulinum toxins, including the dystonias, muscle overactivity in upper motor neuron syndromes/neurologic disorders, sialorrhea, headache, and muscle spasm ( Table 2 ). Clinicians may be less familiar with many of the novel off-label applications for botulinum toxins, which include treatment of disorders affecting a wide variety of body systems and conditions ( Tables 3 and 4 ).

Physicians who provide BoNT treatment must have an extensive knowledge of anatomy, physiology, and biopharmacology. Additional requirements include clinical skills in problem solving, setting realistic goals, determining the correct target for injection, and dose and dilution of toxin. A prerequisite is the skill needed to isolate the target structure during the procedure. This last step, accurate injection technique, is critical for effective treatment as well as minimizing risk and potential adverse events. Techniques used for muscle localization include anatomic guidance/palpation, electromyography (EMG), electrical stimulation (E-stim), and ultrasound. Each of these techniques has well-documented advantages and disadvantages ( Table 5 ). Techniques other than ultrasound have limited value when muscles are not the target for BoNT injection.

Ultrasound Technology for BoNT Injection

Although there are several modes of ultrasound available, B mode is used for live procedural guidance. This mode provides direct visualization of the target structure, the needle, and the toxin entering the target. Under direct visualization the needle can be adjusted to avoid nerves, vessels, and tendons, and to redirect the needle (with a single needle stick) to multiple sites within a target. Ultrasound allows the physician to observe the effect of the injection volume in a given target and to redirect the injection to avoid overloading the muscle or target, which could potentially reduce the risk of spread of the toxin outside the target structures. Ultrasound does not require the active participation or cooperation of the patient. Therefore, is has clear advantages in patients with limited motor control or involuntary movements, and in patients who cannot or will not follow directions or cannot cooperate. Identification of muscles and other targets using ultrasound is based on pattern recognition and identifying structures such as bones and vessels adjacent to the target.

Ultrasound-Guided BoNT Injections for Specific Conditions

Ultrasound for procedural guidance for BoNT injection has been reported for a wide variety of target structures, most commonly muscle, but increasingly in salivary glands and other structures. This section comprises a review of reported uses of ultrasound for BoNT injections in specific conditions and structures. Many of these applications of BoNT are off label.