Botulinum neurotoxins are currently categorized into seven distinct serotypes: A, B, C1, D, E, F, and G.13 The molecules vary in their biosynthesis, size, cellular sites of action, binding kinetics, duration of effect, and stability. The serotypes currently commercially available, serotypes A and B, are derived from different strains of Clostridium botulinum. They both have 150-kDa dichain polypeptides with a heavy chain and light chain linked by disulfide bonds. During biosynthesis, the molecules of A and B can be surrounded by proteins to form a neurotoxin complex, ranging from 500 kDa to 900 kDa (Fig. 255-1). Xeomin® (incobotulinum toxin A) consists of the 150 kDa dichain without accessory proteins.
Schematic of the botulinum complex showing the binding domain with the N-terminus (yellow) and the C-terminus (red), together with the translocation domain (green) and the light chain (blue). (Used with permission from Turton K, Chaddock JA, Acharya KR: Botulinum and tetanus neurotoxins: structure, function and therapeutic utility. Trends Biochem Sci 27:554, 2002.)
The toxin enters the nerves by binding to surface protein receptors and undergoing endocytosis into internalized vesicles. The light chain is released into the nerve cytosol, and the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complex is cleaved to inhibit exocytosis of the neurotransmitters such as acetylcholine (Fig. 255-2). Type A toxin cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), whereas type B cleaves VAMP (vesicle-associated membrane protein), also called synaptobrevin (Fig. 255-3). These proteins are necessary for the release of acetylcholine from vesicles within the cytoplasm of the motor nerve endings. The binding characteristics of each serotype dictate the locus of action on the intracellular SNARE protein complex (Table 255-1).
The heavy chain domain of the botulinum neurotoxin complex binds to the plasma membrane receptor (1) and the complex is internalized (2). The light chain fragment is then released into the cytoplasm (3), where it cleaves the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complex at a site determined by the neurotoxin serotype (4). This disruption of the SNARE complex prevents exocytosis of acetylcholine (ACh) into the synaptic space of the neuromuscular junction. A through G = neurotoxin serotypes; AChR = acetylcholine receptor; LC = light chain; HC = heavy chain C-terminus; HN = heavy chain N-terminus; SNAP-25 = synaptosome-associated protein of 25 kDa; VAMP = vesicle-associated membrane protein. (Used with permission from Turton K, Chaddock JA, Acharya KR: Botulinum and tetanus neurotoxins: Structure, function and therapeutic utility. Trends Biochem Sci 27:555, 2002.)
Schematic representation showing the sites of action of the different botulinum serotypes on the intracellular protein complex known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins responsible for exocytosis of acetylcholine from the nerve. Botulinum A serotype cleaves SNAP-25 (synaptosome-associated protein of 25 kDa). Other serotypes impact VAMP (vesicle-associated membrane protein), synaptobrevin, or syntaxin. BoNT/A through BoNT/G = botulinum neurotoxin serotypes A through G; TeNT = tetanus neurotoxin.
Table 255-1 Binding Sites in the Snare Protein Complex of the Seven Known Botulinum Toxin Serotypes ||Download (.pdf)
Table 255-1 Binding Sites in the Snare Protein Complex of the Seven Known Botulinum Toxin Serotypes
SNAP-25 and syntaxin
The end result is a chemodenervation of the cholinergic neurons, either motor nerves or autonomic nerves, leading to localized absence of skeletal muscle activity or autonomic control of target organs such as the eccrine sweat glands. The way in which the nerves escape the effect of the neurotoxin is partially understood.14 The chemodenervated nerve endings develop collateral sprouting near the primary terminus of the nerve. These sprouts eventually make proximate contact with the targets, either muscle or gland, and begin to overcome the loss of neurotransmitter at the end organ synaptic junctions.
Once these sprouts have reestablished chemical contact with their targets, muscles resume activity and glands begin to secrete. Simultaneously, the original chemodenervated terminal nerve ending begins to degrade the blocked SNARE proteins and develop new proteins to resume the chemical exocytosis of acetylcholine. While these repairs are underway, and while the original terminal nerve ending reacquires communication with the target organ, the collateral sprouts begin to slowly resorb until anatomically nothing is left but the original terminal ending with restored junctional activity.
Four commercially available preparations of botulinum neurotoxin were on the market in the United States and Europe in late 2011, and one was pending approval. All but one are serotype botulinum toxin A, and one is a serotype botulinum toxin B. The products differ in their methods of manufacture, commercial form, and biologic profiles. (Table 255-2).
Table 255-2 Comparison of the Properties of the Commercially Available Forms of Botulinum Toxin in Current Medical Use in the Us (Four) and Outside the United States (Three) ||Download (.pdf)
Table 255-2 Comparison of the Properties of the Commercially Available Forms of Botulinum Toxin in Current Medical Use in the Us (Four) and Outside the United States (Three)
Pending FDA approval
HSA (500 μg)
HSA (125 μg)
HSA (500 μg/ml)
HSA (1 mg/vial)
Sucrose (5 mg/vial)
HSA (500 μg)
Porcine gelatin 5 mg
Dextran 25 mg
Sucrose 25 mg
∼ 900 kD
∼ 500 kD
∼ 700 kD
∼ 150 kD
∼ 150 kD
∼ 150 kD
∼ 500 kD
(units per vial)
per vial (ng/Vial)
∼ 5 (100U vial)
4.35 (500U vial)
0.6 (100U vial)
1 (100U vial)
The units by which these products are described are not interchangeable because of the nature of the assays used to determine their potency. The mouse assays used differ in the diluents used and are not comparable. There is no such thing as a standard neurotoxin unit, hence there is no “International Unit” for neurotoxin. Thus, there is no way of standardizing neurotoxin units to compare a serotype A products to each other, let alone to other serotype B products.
Although the molecule complexes are unique, similar uses are based on clinical observations. For example, in the glabellar frown line pivotal trials, 20 units of Botox® or Xeomin® and 50 units of Dysport® were used.16,17 Botox® is generally diluted with either 1.0 or 2.5 mL of saline per 100 units, producing concentrations of either 10 units per 0.1 mL or 4 units per 0.1 mL respectively. Xeomin® is diluted with 2.5 ml per 100 unit vial. Allergan's pivotal trial used the 4 unit per 0.1 mL dilution. Dysport® is usually diluted with 1.5 mL of saline per 300 unit vial producing a concentration of 10 units in 0.05 mL as used in the pivotal trial. Notice that the Dysport® pivotal trial used injection volumes that were half those used in the Allergan trial: 0.05 mL (10 U) per injection point (Dysport®) versus 0.1 mL (4 U) per injection site (Botox®). It is unclear whether such differences in volume may contribute to behavioral differences between the two products in terms of diffusion and persistence.
The use of sterile saline with preservative (benzyl alcohol) as a diluent appears to lessen the sting of injection with Botox® and Dysport®. Myobloc®, the B serotype, causes more discomfort on injection because of its low pH, but it is stable in liquid form at room temperature for many months.
In addition to the drug name changes, which the FDA required of the manufacturers in 2009, the agency instituted a Risk Evaluation and Mitigation Strategy (REMS), and a boxed warning for these products that warned of the possibility of spread distant from the injection site with potentially life-threatening consequences. Emphasizing the non-interchangeability of these commercial products, the FDA wished to minimize the possibility of medication errors as well as draw attention to the need for tailoring specific doses of each toxin product to specific situations.
The possibility of antibody-mediated resistance appears to be largely theoretical. Original Botox® batch No. 79–11, widely used in ophthalmology and neurology for years, produced rare cases of nonresponse in the treatment of torticollis and blepharospasm. Newer batches introduced in 1997, No. 91223US and No. BCB2024, have significantly less protein load. Although cases of primary nonresponse may be rarely encountered, immunologic resistance to Botox® and Botox Cosmetic® does not appear to be clinically relevant in dermatology, even at the dosages used to treat hyperhidrosis, which can average 400 units per treatment session. In addition, the treatment interval does not appear to be a significant factor in clinical resistance for the newer batches and at the smaller dosages used in cosmetic facial treatment, although exposure to the toxin at increasingly shorter intervals may be associated with development of neutralizing antibodies.