1Pharmacology, immunology, and current developments

INTRODUCTION

Botulinum neurotoxins are proteins synthesized by clostridial bacteria. For clinical use, these proteins are isolated, purified, and formulated into specific products in a complex series of steps that are strictly regulated by governmental agencies in most countries where the products are approved. Because botulinum neurotoxins are derived from living organisms, they are regulated as biological products as opposed to conventional, synthetic drugs. For biological products, the method of manufacture determines not only the purity of the final product but also the reproducibility of unit activity—the dosage measurement for botulinum neurotoxins. The final formulation of the product is also critical, as this can affect product stability, efficacy, safety, and immunogenicity.

This chapter discusses the history, synthesis, and structure of botulinum neurotoxins, as well as their basic and clinical pharmacology. We also review the immunology of these proteins, our understanding of which has deepened in recent years with the identification of specific epitopes against which neutralizing antibodies are formed. Finally, we introduce several current developments in botulinum neurotoxin therapy that have broadened our view of the pharmacology of these proteins, as well as opened new avenues for clinical therapy.

HISTORY

Botulinum toxin type A stands alongside digitalis, atropine, and paclitaxel as natural compounds that, although first noted for their toxic properties, are now routinely used as medicines. The recorded history of botulinum neurotoxins dates back to human encounters with improperly stored food, which caused the sickness known as botulism when ingested (Fig. 1.1). In the early 1800s, the German physician Kerner provided one of the earliest descriptions of food poisoning caused by botulism that followed ingestion of smoked sausages (1). In the late 1800s, Professor van Ermengem, a Belgian microbiologist, identified botulinum neurotoxin as the cause of botulism in a group of Belgian musicians who had eaten inappropriately prepared sausages.

The events of the Second World War stimulated research and study into the activity of botulinum neurotoxins. Much of this research was conducted by Drs. Lamanna, Schantz, and colleagues at Fort Detrick, Maryland, where botulinum neurotoxin type A (BoNTA) was purified, obtained in crystalline form, and synthesized in sufficient quantities for research (Fig. 1.1) (1). A number of other investigators, including Burgen and Brooks, made much progress throughout the late 1940s and 1950s in understanding the mechanism of action of botulinum neurotoxins. By the late 1960s, the inhibitory effects of botulinum toxin type A on acetylcholine release at the neuromuscular junction had been well characterized in experimental animals (1).

Working at the Smith-Kettlewell Eye Research Institute in San Francisco in the 1970s, ophthalmologist Alan Scott was investigating alternatives to surgery for his patients with strabismus, a condition of ocular misalignment. Dr. Scott believed that a substance that could chemically weaken the extraocular muscles pulling the eyes out of alignment might prove a useful alternative to surgical excision of the muscles. On the advice of a colleague, Dr. Scott contacted Professor Edward Schantz (Fig. 1.1) to ask whether he had a substance that might be used to produce such chemical denervation. Schantz suggested botulinum toxin type A and Scott soon reported that this protein was able to correct strabismus in an experimental model (1).

The minute quantities of botulinum toxin type A injected directly into its site of action (in this case, extraocular muscles) prevented systemic absorption of clinically significant amounts.

Following this initial success, Schantz, now working at the University of Wisconsin, began developing botulinum toxin type A for testing in humans for Dr. Scott, focusing on purification, high potency, and preservation. Because no protein drugs of this type had ever been developed, the methods and requirements were novel. Schantz selected the Hall strain of Clostridium botulinum for type A toxin for production because it yielded a good quantity of high-quality toxin, which was necessary for further purification and regulatory requirements. Scott went on to successfully use the botulinum toxin type A that Schantz had produced for the treatment of strabismus and blepharospasm in humans (2). The batch of botulinum toxin type A developed by Schantz was eventually approved for human use by the U.S. Food and Drug Administration (FDA) in 1989 (Fig. 1.1) under the name Oculinum. This preparation was later acquired by Allergan Inc. and, under the name BOTOX®, has been the primary treatment for focal dystonias since the late 1980s, and, over the past decade, has become an important adjunctive treatment worldwide for adult spasticity and juvenile cerebral palsy. The FDA approved the use of BOTOX® Cosmetic in 2002 for the temporary improvement in the appearance of moderate to severe glabellar lines associated with corrugator and/or procerus muscle acidity in patients 18 to 65 years of age.

SYNTHESIS, STRUCTURE, AND PRODUCTS

Botulinum neurotoxins are produced by bacteria as multimeric protein complexes consisting of the neurotoxin and associated hemagglutinin and nonhemagglutinin proteins. These neurotoxin-associated proteins stabilize and protect the 150 kDa type A botulinum neurotoxin from degradation in the gastrointestinal tract, as well as enhance its enzymatic activity (3,4). Different bacterial strains synthesize complexes that vary in size and protein composition, as well as neurotoxin serotype (5).

Seven different botulinum neurotoxin serotypes (A, B, C1, D, E, F, and G) and three different sizes of protein complexes have been reported in the literature. The serotype and protein complex size appear to covary, such that each serotype is associated with a specific set of complex sizes (Table 1.1) (5). All of the serotypes form the 300 kDa complex; serotypes A, B, C1, and D (hemagglutinin positive) form the 500 to 700 kDa complex and only type A forms the 900 kDa complex (6,7).

When botulinum neurotoxin products are manufactured for clinical use, the neurotoxin complexes are isolated and purified using procedures that are specific to the manufacturer. These processes determine which, if any, of the neurotoxin-associated proteins are retained in the final product. For example, during the purification process used to manufacture the Allergan botulinum toxin type A product (henceforth identified as onabotulinumtoxinA), only the900 kDa complex is retained (8,9), whereas in the purification process used to manufacture the Ipsen type A product (henceforth identified as abobotulinumtoxinA), an unknown mixture of complexes are retained (10). All of the neurotoxin-associated proteins are removed in the purification and manufacture of a botulinum neurotoxin type A product Xeomin® (U.S. nonproprietary name assigned as incobotulinumtoxinA) from Merz. This product is currently awaiting FDA approval (as of April 2010) but is available in several

European Union countries (11). The BoNTB-based product, Myobloc®/Neurobloc® is a 700 kDa complex. It has been assigned the chemical name rimabotulinumtoxinB. All approved botulinum toxinbased products are assigned a unit of activity, which are specific for each product and are not interchangeable nor convertible between products.

The active botulinum neurotoxin protein in all serotypes is synthesized as a single chain of approximately 150 kDa that must be nicked or cleaved by proteases in order to be active (Fig. 1.3) (12). Cleavage results in a di-chain molecule consisting of an approximately 100-kDa heavy chain and an approximately 50-kDa light chain, linked by a disulfide bond (5).

The crystal structure of botulinum neurotoxin type A was first reported by Professor Raymond Stevens and colleagues (13), which confirmed many of the predictions made based on studies of physiology and pharmacology. The protein structure is flat and comprises three modules: the endopeptidase (light chain), the translocation domain (N-terminal half of the heavy chain), and the binding domain (C-terminal half of the heavy chain). The crystal structure of botulinum neurotoxin type B (BoNTB) is similar to that of BoNTA (14). Reports on the crystal structures of the light chains of

botulinum neurotoxin serotypes D (BoNTD) and G (BoNTG) have provided insights into the structural details of protease substrate recognition, as described in the next section (15,16).

PHARMACOLOGY

Mechanism of Action

Botulinum neurotoxins exert their activity through a multistep process that includes binding to nerve terminals, internalization, and inhibition of calcium-dependent neurotransmitter release (17). All neurotransmitters are calcium-dependent. This chapter focuses on recent developments in the mechanism of action of botulinum neurotoxins, and the reader is referred to several comprehensive reviews for additional information on the basic mechanisms (18,19).

Binding

The heavy chain ( 100 kDa) subunit of the botulinum neurotoxin molecule binds to receptors on nerve terminal membranes, located primarily but not exclusively on cholinergic neurons (20,21). The binding of botulinum neurotoxins to nerve cell membranes has been explained via a double receptor model, in which the coreceptor

comprises a ganglioside and protein component. Botulinum neurotoxins have long been known to interact with gangliosides (22), with the exception being BoNTD, which appears to bind to a phospholipid but not to gangliosides (23). The crystal structure of BoNTA in complex with the ganglioside cell surface coreceptor GT1b (G = ganglioside; T = trisialo-ganglioside; 1b = carbohydrate’s sequence) has recently been reported (24,25). Based on these observations, the authors suggested that GT1b may mediate the initial contact between the botulinum toxin and the neuronal membrane, which would serve to greatly increase the local toxin concentration at the membrane surface, permitting the toxin to diffuse in the plane of the membrane and bind to its protein receptor (Fig. 1.4) (24).

The protein component of the receptor for botulinum toxin type A has been identified as secretory vesicle protein synaptic vesicle protein 2 (SV2) (26,27). During exocytosis, intravesicular portions of SV2 proteins are exposed to the cytoplasm, providing an exposed surface to which BoNTA can bind (Fig. 1.5) (26,27). Additional research in neuroblastoma cells suggests that FGFR3 (fibroblast growth factor receptor-3) may also be a possible protein receptor for botulinum toxin type A (28). Synaptotagmins I and II have been identified as the protein receptors for BoNTB and BoNTG (29,30). Synaptotagmins are localized to synaptic vesicle membranes and binding of BoNTB and BoNTG to these proteins leads to their internalization into neurons (30,31). The crystal structure of BoNTB in complex with synaptotagmin II has been reported (30,32). The authors observed that synaptotagmin II formed a short helix that bound to a hydrophobic groove in BoNTB and BoNTG; this binding groove varied in other serotypes, supporting the serotype differences in protein co-receptors (30).

Translocation

Following binding, botulinum neurotoxins are translocated into the neuronal cytosol via receptor-mediated endocytosis (33). There appear to be two distinct internalization processes: a rapid uptake, which may utilize the vesicle recycling system, and a slower uptake requiring hours, which may be a less specific endocytotic process. This internalization

process is energy-dependent and is critical for the activity of botulinum neurotoxins (34). Upon acidification of the endosome, it is hypothesized that a pH-dependent change in the translocation domain of the heavy chain facilitates the translocation of the light chain into the cytoplasmic compartment. The exact mechanism of this translocation process is not known, but it has been speculated that the heavy chain can form a pore through which the light chain can pass (33). Recent data indicates that acidification does not trigger substantial structural changes to the botulinum toxin protein as previously thought, but instead may eliminate repulsive electrostatic interactions between the translocation domain and the membrane, leading to the protein’s translocation (35).

(C)

(D)

Figure 1.3 Structure of botulinum neurotoxin unnicked, inactive single-chain protein (150 kDa; left) and nicked, activated di-chain protein (100-kDa and 50-kDa chains; right).

(E)

(F)

Figure 1.4 Potential binding model for botulinum toxin type A. BoNTA displayed as rainbow colored ribbon, GT1b as CPK spheres and Synt-II as a gray ribbon. (A) Free BoNTA above the cell surface displaying GT1b. (B) BoNTA bound to GT1b on the cell surface. (C) BoNTA bound to GT1b and Synt-II on the neuron surface. (D) BoNTA entering the cell through endocytosis. (E) Side view of the BoNTA along the axis of possible rotation. (F) The N-terminal domain of the translocation domain (loops 600 and 760) of the 100 Å long helixes from the translocation domain swinging into contact with the membrane inside the acidified endosome; it is also possible that the other end of the translocation domain make the initial contact with the membrane. Source: From Ref. 24.

Enzymatic Activity

Once inside the cytosol, the light chain cleaves one or more of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins necessary for vesicle docking and fusion, thereby reducing exocytotic neurotransmitter release (Fig. 1.6). Each serotype cleaves a specific peptide bond on one or more of the SNARE proteins (33). The enzymatic activity of the light chain requires the presence of the intramolecular zinc.

In response to reduced neurotransmitter release, neuronal sprouts appear at motor-nerve terminals and nodes of Ranvier, which have been noted within 2 days after injection of BoNTA into mammalian soleus muscles (36). These sprouts persist and become more complex (increased branching and length) for at least 50 days following intramuscular injection of BoNTA. Sprouts may establish functional synaptic contacts (36). However, recent evidence at the rat neuromuscular junction indicates that neurotransmitter release can be detected from

Neuromuscular junction Motor nerve terminus

Muscle cell

Synaptobrevin

SNARE

proteins SNAP-25

Syntaxin

Nerve terminus

Synaptic cleft

Muscle cell

(A)

Exposure to botulinum toxin

Botulinum toxin endocytosed

Normal neurotransmitter release

Vesicle and terminal membranes fuse

Synaptic fusion complex

Light chain cleaves specific SNARE proteins

original terminals about the time that new sprouts have established a functional synapse, and accounts for more than 80% of total acetylcholine release (37). Eventually, exocytosis is restored, the original terminals recover, and the sprouts regress (38). After re-innervation is complete, the target tissue is fully functional (36) and there is no clinical indication that postbotulinum re-innervation produces functionally substandard synapses. However, in rats, acetylcholine release has been found to recover more slowly after multiple injections than

single injections (37). If this finding were also true in humans, it may suggest a tendency for increased duration of clinical response following multiple injections; however, this has been reported in only one study out of 44 studies of repeated injections with BoNTA (39). The dosage did not change in 22 of 44 studies, increased in 4 studies, and were not reported over time in 17 studies (39). The relevance of the preclinical observation to the clinical results remains to be determined.

Lack of Retrograde Transport

Unlike tetanus toxin, the active portion of the botulinum toxin protein does not undergo retrograde transport and transcytosis across neurons to exert effects in distant regions (40). However, Antonucci and colleagues recently published a paper that appears to contradict this wellknown distinction between botulinum and tetanus toxins (41–43). These authors reported retrograde transport and transcytosis of botulinum toxin into rat brain following administration into the muscles that control whisker movements—a report that was rapidly taken up by the popular press (44) and even the Journal of the American Medical Association (45).

However, the results of this study are complicated by a number of important issues (46). First, the authors used a high dose of a laboratory preparation of BoNTA that was injected into a single site of the rat whisker pad (41). The dose used was 135 pg or approximately 450 pg/kg. By way of comparison, patients treated with onabotulinumtoxinA for cosmetic glabellar treatments typically receive approximately 20 units or 3 pg/kg administered into multiple muscles, which is 150-fold lower than the dose used by Antonucci and colleagues. Administration of this high dose may have triggered nonspecific uptake and could have overloaded the protein transport system of the neuron. The use of such high doses of a different type (i.e., laboratory preparation BoNTA) into a single site negates the relevance of these results in a clinical setting with humans. Over 20 years of treatment with BoNTA (onabotulinumtoxin A), of the facial area has not observed any deleterious central effects and that physicians can safely use BoNTA for therapy (47).

A second issue with this study is that the authors used an incompletely characterized antibody to differentiate between cleaved and uncleaved SNAP-25, the key substrate for BoNTA (41). The appearance of cleaved SNAP-25 in central neurons was taken to indicate retrograde transport of the active BoNTA enzyme. That is, the authors did not attempt to directly determine the presence of BoNTA in the central neurons but rather detected a fragment of the protein known to be cleaved by the BoNTA enzyme as a measure of toxin activity in the brain. They detected this protein fragment using Western blot or immunohistochemistry. However, the inference that a positive signal in these assays indicates the presence of cleaved SNAP-25 depends on the specificity of the antibody used. Because the antibody was not well characterized, it cannot be concluded with certainty that the protein binding to it was cleaved SNAP-25. A study that attempted to directly detect botulinum toxin type A in various tissues following intramuscular injection using radiolabeled neurotoxin did not find evidence of distribution into the central nervous system (48).

These points represent major objections to the findings from the Antonucci et al. study. The conclusions of the study do not appear to be entirely justified and, in particular, their relevance to clinical use of botulinum neurotoxins is questionable.

Nonmotor Anticholinergic Effects

Botulinum neurotoxins act not only on efferent motor pathways but also on efferent autonomic efferent pathways, which also utilize acetylcholine as a neurotransmitter. The inhibitory effects of BoNTA on autonomic nerve terminals have led to its successful use in conditions of autonomic hyperactivity such as hyperhidrosis and gustatory sweating (49). Although the effects on autonomic and motor nerve terminals are thought to occur by a similar mechanism (i.e., binding, internalization, and inhibition of neurotransmitter release), the clinical effects are of longer duration in some autonomic conditions than in neuromuscular conditions. The reason for this difference is unknown.

Direct evidence from preclinical studies and indirect evidence from clinical studies indicate that BoNTA affects afferent pathways via inhibition of neural input to intrafusal fibers (49,50). Intrafusal fibers are encapsulated fibers that make up muscle spindles (Fig. 1.7), or the

proprioceptive organs located among skeletal muscle fibers (extrafusal fibers). Extrafusal fibers are innervated by alpha motor neurons, whereas intrafusal fibers are innervated by gamma motor neurons and Ia sensory afferents. The inhibition of gamma motor neurons decreases activation of muscle spindles, which effectively changes the sensory afferent system by reducing the Ia traffic. Filippi and colleagues confirmed this hypothesis by establishing that local injections of BoNTA directly reduce afferent Ia fiber traffic in rats, thereby modulating sensory feedback (50). Histologic support for the direct effect of BoNTA on the rat muscle spindles supported the electrophysiologic results (51).

Berardelli and colleagues have examined potential clinical correlates of BoNTA’s effects on intrafusal fibers. These investigators evaluated electrophysiological variables in muscles injected for the treatment of writer’s cramp (52). In these patients, BoNTA reduced the tonic vibration reflex to an even greater extent than the maximal M-wave and maximal voluntary contraction. Additionally, the tonic vibration reflex but not the other variables remained reduced in several patients whose clinical benefit persisted for 7 months. The authors speculated that suppression of the tonic vibration reflex may result from effects of the neurotoxin on intrafusal muscle fibers, causing reduced spindle inflow to the central nervous system during vibration (52). Similar effects were noted in individuals with spasticity who retained some degree of motor function (53). Combined with the preclinical findings, these results suggest that the overall effect of BoNTA therapy, at medically relevant doses, may be a combination of a direct effect on the primary nerve-end organ communication coupled with an indirect effect on the overall system. These reports utilized onobotulinumtoxinA and should not be applied to other products, as summarized below.

Research documenting the effects of botulinum neurotoxins on neurotransmitters other than acetylcholine is accumulating, particularly as it relates to pain and urinary tract dysfunction. The current developments in this area are discussed in the last section of this chapter.

CLINICAL PHARMACOLOGY

Differences Between Botulinum Neurotoxin Products

Because botulinum neurotoxins are biological products, their clinical pharmacology depends on a variety of factors, including the bacterial strain used in production, methods of isolation and purification, serotype, formulation, and procedures used to determine biological activity (Table 1.2). These factors vary for each commercially available botulinum neurotoxin product. Each product’s distinct formulation results in a unique interaction with biologic systems following injection. The system is exposed to different ingredients and different numbers of molecules that likely influence local osmotic gradients and diffusion. Additionally, isolation and purification methods can influence the antigenicity of biological products (54). Even minor changes to the formulation of biological products can influence clinical profile, as demonstrated with a human erythropoietin analog epoetin alfa (54). In Europe, the switch from albumin to polysorbate 80 in the formulation of one of these products [Eprex (J&J), in EU, Australia,

Figure 1.7 Muscle spindle structure showing intrafusal and extrafusal fibers.

Singapore, and Canada] led to an unpredicted increase in cases of pure red cell aplasia—a severe form of potentially lethal anemia (54–57). Additional changes to the product have since reduced cases of this normally rare immunogenic response to low levels (55), but this example illustrates the complexity of biological products and the unpredictable effects of even small changes in manufacturing process or formulation.

Of paramount importance with botulinum neurotoxin products is the difference in units of biological activity. Units of different botulinum neurotoxin products are not equivalent and cannot be interchanged using a single ratio (58,59). Not only do botulinum neurotoxin products exhibit pharmacologic differences (60,61), but they are also used clinically at different doses depending on the indication and individual presentation (62).

Approved Products

Even products that are labeled as containing the same number of units per vial do not necessarily exhibit the same biological activity. This was demonstrated in a recent comparison of two BoNTA products, both labelled at 100 units (63). One of the products botulinum neurotoxin A (incobotulinumtoxinA, Merz) was found to contain substantially fewer units per vial when compared against onabotulinumtoxinA reference standard (63). Additionally, units of the incobotulinumtoxinA product were substantially lower when tested one year later, which was still prior to the product’s expiration date. These results suggest product degradation over time and emphasize that attempts to interchange BoNTA products fail to take into consideration potentially critical effects of formulation on biological product performance.

Many attempts have been made to compare the units of several established BONTA products (onabotulinumtoxinA and abobotulinumtoxinA) (58,64,65). Although in the past it was reported by some investigators that these two products were clinically comparable

Table 1.2 Characteristics and Packaging of Different Botulinum Neurotoxin Products (11,59,108,109)

when used at dose ratios of approximately 1:3 to 1:5, a growing body of evidence suggests that the products exhibit different clinical characteristics regardless of the dose ratio (58,66,67). In particular, abobotulinumtoxinA seems to exhibit a somewhat different side effect profile than onabotulinumtoxinA (66,68–70). This conclusion is also supported by preclinical comparisons, which are more highly controlled and can employ a broader range of doses than is possible in human studies (60,61). Despite these results, some authors report dose ratios as low as 1:1 (71). It should be noted that the bulk of the literature does not support this dose ratio, and it does not represent clinical practice (62). Clinical use of this dose ratio could have serious consequences for patients; for instance, use of onabotulinumtoxinA at the higher doses needed for abobotulinumtoxinA could lead to inadvertent side effects, whereas use of abobotulinumtoxinA at doses used for onabotulinumtoxinA could lead to inadequate efficacy and duration of action. Clearly, to maximize patient safety and clinical benefit, it is critical that clinicians use each BoNTA product at doses that have been established for that specific product in the specific indication.

The botulinum toxin type B (rimabotulinutoxinB, BoNTB) from Elan/Solstice also cannot be compared to other products based on a dose ratio. Doses of this product are often up to several orders of magnitude higher than onabotulinumtoxinA depending on the indication and individual patient presentation, and adverse event profiles differ (72).

Unlicensed Products

The case against dose conversion of botulinum neurotoxins has become even more prominent with the unscrupulous use of counterfeit and unlicensed products. One case of nonequivalent units was demonstrated with a botulinum toxin type A product CNBTX-A (Nanfeng) that was previously available in China but was not approved there or in any other country (73). The label on each vial indicated 55 units, but the product was not accompanied by a package insert or dosing recommendations. Testing against an onabotulinumtoxinA reference standard showed that a vial of CNBTX-A contained 243 units of biological activity (73). Serious consequences could have resulted if clinicians had obtained this nonapproved product and applied it to patients based on doses of an approved product. This alarming variation in biological activity strongly indicates that clinicians must not rely on dosing of one neurotoxin product to ascertain dosing of another. This finding also indicates the dangers of nonapproved neurotoxins—the lack of literature to guide dosing of this product and the lack of an approved manufacturing process could lead to serious, unintended consequences for patients.

These risks were validated by the unscrupulous use of a highly concentrated laboratory preparation of BoNTA (that occurred in 2006). This neurotoxin product, which was clearly labeled for laboratory use only, was illegally administered to several individuals in a Florida clinic for cosmetic purposes (74). All of the individuals exposed to this highly concentrated laboratory preparation of BoNTA experienced progressive muscle weakness and neuropathies, and were eventually hospitalized for up to 14 weeks (74).

The dangers of using unlicensed botulinum neurotoxin preparations are unambiguous: clinicians risk patient safety. It is critical that clinicians verify the botulinum neurotoxin product they are using and use it at doses recommended by the manufacturer and/or documented in the published clinical literature. The data documenting the nonequivalence of botulinum neurotoxin units is summarized in Figure 1.8.

Neuromuscular Injection

In the clinic, BoNTA is most often injected into overactive muscles that vary depending on the condition to be treated and the patient’s individual presentation. Onset of action following intramuscular

injection is approximately 3 to 7 days (75). The beneficial effects of each treatment with BoNTA last approximately 3 to 5 months in neuromuscular conditions (75,76). The duration of BoNTB is somewhat shorter than that of BoNTA and has been reported as 6 to 8 weeks with 1000 units and 10 to 12 weeks with 2000 units in the management of brow or glabellar lines (75).

Due to the chronic nature of most of the neuromuscular conditions that botulinum neurotoxins are used to treat, repeated injections are typically required over the course of many years. The results of numerous studies indicate that most patients respond to BoNTA for many years without decrements in safety, responsiveness, or quality of life, and without increased doses (77,78). Some studies have reported enhanced benefits with BoNTA following repeated injections, showing increased duration, decreased adverse events, or greater functional improvements (e.g., gait in children with cerebral palsy) with successive injections (39). In the case of improved gait, this may be due to adaptation of the patient to reduced tone. However, the increased duration and other benefits may also be due to altered sensory feedback from the periphery to the central nervous system (79).

Intradermal Injection

In the treatment of focal hyperhidrosis, BoNTA is injected intradermally instead of intramuscularly. The onset of action of BoNTA in various forms of hyperhidrosis is within 1 week, and benefits last approximately 7 months (80,81). Benefits are maintained following repeated injections for at least 16 months (80).

Several studies have examined the use of BoNTB (rimabotulinumtoxinB) for axillary hyperhidrosis. These studies have found that type B significantly reduces sweating, but with distal autonomic side effects that are not observed with type A such as visual accommodation difficulties and dry mouth (82).

IMMUNOLOGY

Like most foreign proteins introduced into the body, botulinum neurotoxins can be antigenic and, under the certain circumstances (e.g. dose and frequency), elicit immune responses designed to inactivate the protein. Only antibodies directed against the 150-kDa neurotoxin neutralize its activity (83). Antibodies may occasionally be formed

Approved, licensed preparations

against the nontoxin proteins in the botulinum neurotoxin complex, but these do not affect clinical responsiveness (83).

Within the BoNTA molecule, antibodies directed against certain peptides within amino acid residues 449 to 1296 of the heavy chain are neutralizing (83). Nearly all of the regions overlap or coincide with the regions on the protein that bind to synaptosomes (84), providing a physical basis for their neutralizing effect (i.e., blocking the binding of BoNTA to the nerve terminal). Similar results have been found for BoNTB (rimabotulinumtoxinB) (85). Research has further shown that the pattern of antibody recognition varies among patients with neutralizing antibodies, such that not all patients develop antibodies to the same portion of the BoNTA molecule (84). These findings underscore the role of individual genetic factors in neutralizing antibody development.

In addition to individual genetic factors, manufacturing methods and formulation are known to affect the immunogenicity of biological products (54,86), and thus, it cannot be assumed that the rate of neutralizing antibody formation will be the same with all botulinum neurotoxin products. Both short-term and long-term (e.g. 2-year) studies are needed with each individual product to adequately determine its antigenicity in a given clinical population at relevant doses.

Few studies have been published on the antigenicity of botulinum neurotoxin preparations in cosmetic use, partly because the relatively low doses utilized minimize the potential for neutralizing antibody formation. In a short-term spasticity study and a long-term cervical dystonia study, the rate of neutralizing antibody formation with onabotulinumtoxinA (Allergan) has been documented at approximately 1% (87,88). In these studies, the sera of all available patients were analyzed using the mouse protection assay, which is the gold standard test due to its specificity, despite its relative lack of sensitivity. AbobotulinumtoxinA (Ipsen) did not elicit any antibody formation in a shortterm spasticity study (89); however, in a longer-term study of 93 dystonia patients who received a mean of 4 treatments (range 1–13), the overall rate of neutralizing antibody formation was 3% (4% among cervical dystonia patients) (90). The neutralizing antibody formation rate with incobotulinumtoxinA (Merz) has not been reported. The neutralizing antibody formation rate with rimabotulinumtoxintyptB (Solstice) has been reported as 10% after 1 year or 18% after 18 months of treatment for cervical dystonia (91).

Each approved

product should be

used at doses

recommended by

the manufacturer or

documented in the clinical literature

Primary or secondary clinical nonresponsiveness has been reported in the absence of neutralizing antibodies, suggesting that there may be other reasons for lack of response to botulinum neurotoxins. These reasons include patient perception (e.g., subsequent injections may appear to have a less dramatic effect than the first) (92), either because patients continue to experience some benefit from the previous injection or perhaps due to lack of memory about the severity of their condition prior to injection. The injections may not be directed into the optimal muscles or the muscles involved may have changed from the previous visit either due to progression of the disorder or neural adaptation (93). These changes may require a modification of injection sites, dose, or both in order to maintain optimal treatment benefit.

CURRENT DEVELOPMENTS

Several interesting developments are currently taking place in botulinum neurotoxin therapy. The first concerns the activity of these proteins in the treatment of pain and the second is the novel information on mechanism of action and clinical benefit derived from the application of these proteins on urinary tract disorders such as overactive bladder and benign prostatic hypertrophy.

Pain

Beneficial effects of BoNTA on pain were stimulated by the finding that injections into patients with cervical dystonia relieved not only the aberrant muscle activity, but also the associated neck and shoulder pain (94). The benefits reported for BoNTA in chronic migraine (95) and the lack of direct concordance between its effects on muscle relaxation and improvement in pain in neuromuscular conditions (96) suggest that pain relief may not be strictly secondary to the reduction of muscle contractions. This has led to an increase in research directed at identifying possible mechanisms by which BoNTA may act to reduce pain.

Pain is transmitted to the central nervous system by two types of afferent nerves or primary nociceptive afferents: A-delta fibers that mediate sharp, pricking pain and C fibers that mediate slow, burning pain. The cell bodies of these neurons are located in the dorsal root ganglia, where they send out a single process that branches to innervate the periphery as free nerve endings (nociceptors—pain sensory organs) and the other to innervate the central nervous system, synapsing on neurons in the dorsal horn of the spinal cord. Pain sensations detected in the face and head are transmitted by trigeminal neurons (A delta and

C fibers) whose cell bodies are located in the trigeminal ganglion and whose axons synapse in the brain stem. Type C fibers release substance P, somatostatin, and other neuropeptides from both central and peripheral terminals. These peptides mediate pain and inflammatory reactions.

BoNTA has been found to inhibit substance P release from cultured dorsal root ganglion neurons (97). Substance P is a peptide neurotransmitter released by primary nociceptive afferents (C fibers). Additionally, botulinum toxin type A has been found to reduce the stimulated but not basal release of calcitonin gene-related peptide (CGRP) from cultured trigeminal ganglia neurons (98). CGRP is an inflammatory neuropeptide that is contained within dorsal root ganglia neurons and colocalized with substance P in most trigeminal and other sensory ganglia neurons. BoNTA inhibits the release of acetylcholine from both alpha and gamma motor neurons, thus eliciting muscle relaxation. Additionally, BoNTA has antinociceptive effects in several animal models (99,100). A possible mechanism by which BoNTA may act in reducing pain through inhibition of pain neuropeptides, direct reduction of peripheral sensitization of the pain nerve, and therefore indirect reduction of central sensitization associated with chronic pain (Fig. 1.9).

Beneficial effects of BoNTA on pain associated with postherpetic neuralgia was reported in several case studies in 2002 (101). A recent randomized, controlled study has confirmed these effects in patients with neuropathic pain due to postherpetic neuralgia or focal nerve injury accompanied by spontaneous pain and mechanical allodynia (Fig. 1.10) (102). Results of this study indicated that BoNTA was more effective than placebo at reducing spontaneous pain intensity, which correlated with the preservation of thermal sensation at baseline (102). BoNTA also improved allodynia and decreased cold pain threshold, without affecting perception thresholds. Neuropathic symptoms and general activity also significantly improved in these patients, and injections were well tolerated. The authors concluded that BoNTA may induce direct analgesic effects in patients with chronic neuropathic pain independent of its effects on muscle tone. These findings suggest that BoNTA may be a useful treatment for certain types of neuropathic pain that include a neurogenic inflammatory mechanism. Some chronic pain conditions include postherpetic neuralgia, painful diabetic neuropathy, complex regional pain syndrome, chronic migraine, overactive bladder (see below), arthritis, etc. Studies examining the effects of these proteins in other conditions characterized by a prominent pain component are ongoing.

P2X3

SP

ATP/ACh

M2

P2X3

M2

NK-1

Ach

M2

M3

Lower Urinary Tract Disorders

BoNTA is an effective therapy for the treatment of overactive bladder, as demonstrated by a recent European consensus report that concluded that “the use of botulinum neurotoxin type A (BoNTA) is recommended in the treatment of intractable symptoms of neurogenic

detrusor overactivity (NDO) or idiopathic detrusor overactivity (IDO) in adults (grade A)” (103).

When injected into the bladder, BoNTA reduces acetylcholine release from parasympathetic cholinergic fibers that innervate the detrusor muscle, leading to muscle relaxation. In addition to these motor effects,

BoNTA may affect sensory nerves in the bladder. In a preclinical model of bladder pain, BoNTA has been found to reduce the release of CGRP from afferent nerves (104). Additionally, BoNTA has been found to affect the vanilloid receptor transient receptor potential vanilloid receptor-1 (TRPV1) and the purinergic receptor P2X3 (105). TRPV1 is expressed on peripheral nociceptors, where increased levels are involved in maintaining inflammatory hyperalgesia. P2X3 (purinergic receptor P2X, ligand-gated ion channel, 3), a receptor for adenosine triphosphate (ATP), is expressed on primary afferent fibers and functions in pain transmission. In individuals with neurogenic or idiopathic detrusor overactivity, levels of TRPV1 and P2X3 receptors were significantly decreased following BoNTA administration, which correlated with improvements in clinical and urodynamic parameters (105). These findings suggest that BoNTA may produce its clinical benefit, in part, by decreasing the levels of these sensory receptors (Fig. 1.11).

BoNTA also reduces the level of nerve growth factor in the bladder of individuals with detrusor overactivity (106). Recent evidence has confirmed that patients with detrusor overactivity exhibit increased levels of nerve growth factor in the bladder, and the reduction of these levels is associated with clinical response to BoNTA (107).

SUMMARY AND CONCLUSIONS

Basic and clinical research on botulinum neurotoxins continues to progress rapidly. The past few years have seen major advances in our understanding of botulinum neurotoxin binding, with the identification of protein coreceptors for serotypes A and B. Our understanding of the immunology of botulinum neurotoxins is also proceeding, as demonstrated by the identification of the epitope regions on the protein’s heavy chain. In addition to the traditional anticholinergic action of botulinum neurotoxins, novel pharmacological actions of these proteins have been identified that may contribute to their mechanism of action in various conditions. These novel mechanisms largely involve sensory afferent neurons and may play a role in botulinum neurotoxin’s effects in primary pain conditions and lower urinary tract disorders.

With the development of additional clinical indications for botulinum neurotoxins, more clinical products based on these proteins are entering the marketplace. Because botulinum neurotoxins are biological products, different preparations manufactured using different methods, and having different formulations possess unique clinical performance profiles. Particularly important to clinicians are the dosing differences among these products. Dose confusion could compromise clinical efficacy or patient safety or both.

As research and development of botulinum neurotoxins advances, it seems likely that additional applications will be identified for these important therapeutic proteins. As in the case of chronic pain and lower urinary tract diseases, investigations into the mechanism of action of botulinum neurotoxins may even expand our knowledge of the disease mechanisms themselves. In such ways, the clinical and basic sciences of botulinum neurotoxins are intertwined.

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