Key points

Background

Aesthetic injection botulinum toxin (BoNT) is the most common cosmetic procedure performed in the world today. BoNT has proven to be one of the most versatile agents used in the aesthetic field, and has also been used for numerous medical applications, including arm and leg spasticity, blepharospasm, cervical dystonia, strabismus, severe hyperhidrosis, chronic migraine, and overactive bladder, to name a few.

Although the only U.S. Food and Drug Administration (FDA)–approved cosmetic indication for BoNT is the treatment of dynamic rhytides in the glabellar area, successful off-label uses of the products have been vast. Currently, the aesthetic market has multiple formulations available, and with the expected availability of more in the near future, it is important to understand that each agent cannot be equivalent and, that differences and similarities exist between them. All products are synthesized by the same strain of anaerobic Clostridium botulinum , a gram-positive, rod-shaped anaerobic bacterium. The therapeutic preparations of botulinum neurotoxin however, vary in molecular weight, complexing proteins and excipients.

Background

Aesthetic injection botulinum toxin (BoNT) is the most common cosmetic procedure performed in the world today. BoNT has proven to be one of the most versatile agents used in the aesthetic field, and has also been used for numerous medical applications, including arm and leg spasticity, blepharospasm, cervical dystonia, strabismus, severe hyperhidrosis, chronic migraine, and overactive bladder, to name a few.

Although the only U.S. Food and Drug Administration (FDA)–approved cosmetic indication for BoNT is the treatment of dynamic rhytides in the glabellar area, successful off-label uses of the products have been vast. Currently, the aesthetic market has multiple formulations available, and with the expected availability of more in the near future, it is important to understand that each agent cannot be equivalent and, that differences and similarities exist between them. All products are synthesized by the same strain of anaerobic Clostridium botulinum , a gram-positive, rod-shaped anaerobic bacterium. The therapeutic preparations of botulinum neurotoxin however, vary in molecular weight, complexing proteins and excipients.

History of BoNT

C botulinum was discovered by professor of bacteriology, Emile Pierre van Ermengem, in 1895, after a botulism outbreak in a Belgian village. The toxin itself, however, was not isolated until the 1920s. Purification of the toxin was attempted by Dr Herman Sommer and colleagues at the University of California, San Francisco, but these attempts were not successful until Dr Edward Schantz isolated a purified BoNT type A (BoNTA) in a crystalline form in the 1940s.

Approximately 30 years later, Dr Alan Scott, a surgeon at the Kettlewell Eye Research Institute in San Francisco, began testing BoNTA in monkeys as a potential therapy for strabismus. After successful treatments on monkeys, Dr Scott performed the first large-scale clinical trial using onabotulinumtoxinA (BoNTA-Ona) (Oculinum) to treat strabismus. It was during this time that Dr Jean Carruthers noticed what was to become the future of BoNTA for aesthetic use: diminishing of wrinkles in the glabellar area. After Dr Scott’s successful use of Oculinum, Allergan licensed his technology, named their product Botox, and received FDA approvals for both therapeutic and cosmetic indications, including strabismus and blepharospasm in 1989, cervical dystonia in 2000, glabellar lines in 2002, axillary hyperhidrosis in 2004, upper limb spasticity and chronic migraines in 2010, and urinary incontinence in 2011. BoNT was the first microbial protein injection used to treat human disease. In 1991, Speywood (now called Ispen) launched another BoNTA formulation based on abobotulinumtoxinA (BoNTA-abo), Dysport, for the treatment of blepharospasm, hemifacial spasm, and focal spasticity. Dysport was then approved for the treatment of glabellar lines in 2009. In 2000, Elan (now Solstice Neurosciences) brought its rimabotulinumtoxinB (BoNTB-rima)-based BoNT type B product Myobloc to market for the treatment of cervical dystonia, currently its only FDA-approved use. More recently, Merz Pharmaceuticals received regulatory approval for Xeomin, a product based on incobotulinumtoxinA (BoNTA-inco) for the treatment of focal dystonias, spasticity, and glabellar lines.

Structure and mechanism of action

Seven serologically distinct BoNTs (BoNT types A, B, C, D, E, F, and G) are produced by various strains of C botulinum , C butyricum , and C baratii in nature. Only serotypes A and B are used therapeutically, with type A being the most neurotoxic. The nontoxic accessory proteins are composed of hemagglutinin and nontoxin, nonhemagglutinin proteins, and spontaneously associate with the core neurotoxin after their cosynthesis by the bacteria.

The size of the neurotoxin complexes is determined by serotype and accessory protein content. For example, type A clostridial strains produce complex sizes of 300, 500, and 900 kDa, whereas type B strains produce complexes of only 300 and 500 kDa.

BoNT consists of 2 protein chains: a heavy chain (100 kDa) and a light chain (50 kDa), connected by a single disulfide bridge and noncovalent bonds. The heavy chain is made up of the binding and translocation domains. The light chain is the actual toxin itself. The binding domain attaches to the receptor on the neuronal end plate. The translocation domain causes endocytosis of the BoNT into the cytoplasm of the nerve. Simply, BoNT exerts its effects by inhibiting acetylcholine (Ach) release at the neuromuscular junction and autonomic nerve terminals. The effects of BoNT are nonpermanent. All formulations of BoNT inhibit exocytosis and Ach release without affecting Ach production or conduction of signaling along the nerve fiber.

The process through which BoNT exerts its effect is dependent on a series of steps that begins with uptake of the toxin into the nerve terminal. BoNTA-ona, BoNTA-inco, and BoNTA-abo bind to the same cellular receptor synaptic vesicle protein 2 (SV-2), whereas BoNTB-rima binds to synaptotagmin II (Syt II) with great selectivity for these glycoproteins at the neuromuscular junction. Once internalized in the cell, the light chain dissociates from the heavy chain and cleaves one or more of the soluble N -ethylmaleimide–sensitive factor attachment receptors (SNAREs), which are part of the Ach transportation cascade and are required for docking of vesicles with the neural cell membrane in the release of Ach from the nerve terminal. The light chain of each serotype of BoNT cleaves a different peptide bond, and no 2 serotypes do so at the same site. The substrate for BoNTA is a 25-kDa synaptosomal-associated protein (SNAP-25). BoNTB targets and cleaves vesicle-associated membrane proteins (synaptobrevins).

Once the neuromuscular junction has been blocked by BoNT, binding sites for the toxin are decreased. As a result, repeated injection into an already denervated muscle is not as effective, because uptake into muscle is reduced. The neurotoxic effect of the neuromuscular junction is temporary. After injection, the effect is evident within 1 to 3 days. Maximal effect is seen approximately 2 weeks after the toxin is introduced, and then gradually starts to decline after approximately 3 months. Restoration of the neuromuscular junction is seen at 2 levels. Axonal sprouting is a temporary recovery process, wherein new neuromuscular junctions develop around the blocked terminal. Once the original neuromuscular junction is restored, the sprouts are removed.

This inhibitory mechanism of action is also used in the treatment of hyperhidrosis. In addition to their effect on the neuromuscular junction, BoNTs have the ability to act on the innervated eccrine secretory cells. Through inhibiting the sympathetic fibers to sweat glands, where Ach is used as the neurotransmitter, BoNTs have been shown to effectively reduce sweat production.

Other indications for the use of BoNT are based on the fact that sensory nerves release mediators of pain and inflammation, including substance P and cGRP (calcitonin gene-related peptide). Vesicles located at sensory nerve terminals also contain SNARE proteins that facilitate the release of these substances. In studies conducted using animals, BoNTA-ona has been shown to reduce cGRP. It has been suggested that blocking cGRP, glutamine, and substance P inhibits neurogenic inflammation and can ameliorate pain syndromes, such as migraine headache or psoriatic skin lesions.

Noninterchangeability of different formulations

All BoNTA products cannot be generics, and each has a unique manufacturing process. BoNTA-ona, BoNTA-inco and BoNTA-abo are derived from a strain of C botulinum type A strain called Hall strain . BoNTB-rima is derived from a type B strain called Bean strain . BoNTA-ona is produced using a crystallization process that yields a homogenous formulation of 900-kDa complexes, whereas BoNTA-abo and BoNTB-rima are both manufactured with column chromatography. The process of BoNTA-abo production yields a heterogeneous formulation of 300- and 500-kDa complexes, whereas BoNTB-rima manufacturing produces a homogenous mixture of complexes. The production of BoNTA-inco is distinctive, because the complexing proteins are separated and discarded from the neurotoxin during the manufacturing process in several purification steps. The resulting pure neurotoxin has a molecular weight of only 150 kDa. The excipient components of the vials, intentionally added substances that do not exert a therapeutic effect but may affect product delivery, vary among the formulations.

All the products contain human serum albumin. Human serum albumin acts as a stabilizer of BoNT. As discussed by Pickett and Perrew, the toxin is protected by the accessory proteins, which must dissociate for the core toxin to act. This dissociation occurs under physiologic conditions, including when the toxins are diluted with saline. Thus, the core toxin may be dissociated from the accessory proteins before injection. The amount of human serum albumin differs between the formulations.

The pH of the formulations depends on the pH of the preserved saline used and the ability of the excipients to buffer the solution. The number of units and the amount of core neurotoxin differs between the formulations. BoNTA-ona and BoNTA-inco are distributed in 100-U vials, BoNTA-abo in 300- and 500-U vials and BoNTB-rima in 2500-, 5000-, and 10,000-U vials. Therefore, the amount of neurotoxin injected depends on the dose of the product used ( Table 1 ).

As a result of the differences discussed, among others, it has been mandated that the product labeling include specific language that states that the unit and dose of each product are not interchangeable and cannot be simply converted between products.

Immunogenicity

Because BoNT is a protein-based drug that is administered repeatedly for long-term effect, it is capable of triggering immunologic reactions and antibody formation. Various factors have an impact on its immunogenicity, such as prior exposure, manufacturing process, antigenic protein load, overall toxin dose administered, and presence of accessory proteins.

Immunity in patients treated for indications requiring high doses, such as cervical dystonia, was as high as 20%. In 1998 the manufacturer improved the purity of the BoNTA-ona formulation to decrease the amount of protein accompanying the toxin. Through decreasing the antigenic exposure, the immunity rates with repeated injections for cervical dystonia decreased and the purification did not alter dosing requirements.

Because the protein content of BoNTA-inco is less than that of BoNTA-ona when comparing a 1-to-1 U dose equivalence, BoNTA-inco is expected to have a lower risk of immunogenicity. Although BoNTA-abo has a 2.5:1.0 equivalence ratio versus BoNTA-ona, the protein load is considerably smaller. Therefore, BoNTA-abo would also have a lower immunogenicity.

However, currently no studies have directly compared antigenicity rates between currently available products. Factors that are thought to increase the incidence of antigenicity include increased treatment number and cumulative dose, frequent exposure, and genetic predisposition. With these risk factors in mind, and given the increased doses used with BoNTA-abo and the shorter dosing interval, one might expect higher rates of antigenicity because of greater exposure to the toxin than when using BoNTA-ona. However, thus far, the immunogenicity rates of BoNTA-ona and BoNTA-inco have been around 1%, whereas the immunogenicity rate for BoNTA-abo is slightly higher at 3%. BoNTA-inco has also been shown to induce an immunogenic response, which was established during the clinical trials performed in the United States. Twelve subjects developed neutralizing antibodies during the course of their trials. The purification process for each BoNTA is different, and therefore experience with BoNTA-ona and immunogenicity cannot be used to estimate the risk of immunogenicity with BoNTA-inco or BoNTA-abo. Overall, BoNTA products exhibit low clinically detectable levels of antibodies when compared with other approved biologic products, especially when used in low doses for aesthetic indications.

Contraindications and adverse events

Although generally treatments with BoNTA are considered safe, practitioners must be aware of the contraindications and adverse events and take the appropriate measures to minimize the risk. BoNT is contraindicated in any patient with known hypersensitivity to BoNT or any component of the formulation and in patients who have an active infection at the site to be injected. Relative contraindications are in patients who are pregnant or nursing and anyone with a preexisting neuromuscular disease, such as myasthenia gravis and Eaton-Lambert syndrome. Although BoNT is classified as pregnancy category C in the United States, no teratogenicity has been reported as a result of women who inadvertently received up to 300 U of BoNTA during pregnancy.

Common adverse events seen in the aesthetic use of the toxin include swelling, localized bruising, headaches, injection site discomfort, excessive muscle weakness, and unintended paresis of adjacent muscles. Most of these adverse events have been comparable to those seen with placebo, as shown in several studies. Avoiding anticoagulants or other blood-thinning supplements 2 weeks before the treatment can reduce the risk of bruising. Injection site discomfort can be avoided by using small-gauge needles and careful technique. If necessary, a topical anesthetic can be used or ice can be applied before the treatment. To avoid unintended muscle weakness, therapy should be initiated with lower doses and titrated upwards to achieve the desired effect. Lastly, using diluents containing preservatives has been shown to reduce patient discomfort during the injection.

BoNT is among the most potent toxins in existence. Safety measures should be known and adhered to by all physicians, regardless of their experience. Proper patient selection and evaluation are crucial, along with precise injection technique. In most cases, adhering to the safety measures will avoid systemic exposure of the toxin.

Handling and storage

Studies have shown that BoNT formulations are not as fragile as suggested in the package instructions. Prescribing information for BoNTA-ona, BoNTA-abo, BoNTA-inco, and BoNTB-rima state that each vial is for single use. BoNTB-rima is available in a ready-to-use form with no reconstitution necessary. Handling instructions state that vials should be refrigerated and used within 4 hours of reconstitution in the case of BoNTA-abo and BoNTB-rima, and within 24 hours of reconstitution in the case of BoNTA-ona and BoNTA-inco. BoNTA-inco, however, does not require refrigeration. Exposure to direct sunlight is thought to inactivate the toxin within 1 to 3 hours, and temperatures of 80°C to 100°C denature the toxin in approximately 30 and 10 minutes, respectively.

Hexsel and colleagues published a study that showed that the efficacy of BoNT was not compromised for up to 6 weeks after reconstitution. Similarly, a different study showed no contrast in the efficacy of freshly reconstituted BoNTA and BoNTA-ona that was reconstituted 2 weeks before being injected and then stored in either a freezer or a refrigerator. This study, and also one conducted by Sloop and colleagues, showed that proper storage could be in either a freezer or a refrigerator.

A recent animal study examined the effect of sustained agitation of BoNTA-ona for 6 weeks after reconstitution. The agitated BoNTA-ona was subsequently injected into mice to determine if it was still lethal (LD50). The study found no loss of efficacy in the vigorously agitated BoNTA-ona. Additional studies have compared the efficacy of gently and vigorously reconstituted BoNTA-ona used for the treatment of rhytides in human subjects. These studies concluded that no loss of effect occurred with vigorous reconstitution.

Onset and duration

Awareness with respect to expected time to relapse of a treated area after BoNT injections is vital in scheduling the subsequent appointment. It is generally accepted that the average duration of effect is between 3 and 4 months. A study conducted by Carruthers and colleagues evaluated the efficacy of BoNTA-ona for the treatment of glabellar lines. The investigators determined that the rate of response at maximal frown lasted 3 months for most patients, and as long as 4 months in 25% of the patients, showing that 3- to 4-month intervals between injections is appropriate. Likewise, a study using BoNTA-abo with a 50-U dose versus placebo determined that the optimal timing for a second injection was between 3 and 4 months. Another study with more than 700 subjects treated with BoNTA-abo found a median duration of effect of 85 to 109 days. One study showed a difference in response rate at 16 weeks between 2 different formulations; the relapse rate was 23% for BoNTA-ona and 40% for BoNTA-abo. BoNTA-inco has been shown to have similar efficacy duration as BoNTA-ona, although more clinical studies are warranted for further comparison.

A recent study of 45 patients using both patient and physician assessments of response after treatment of glabellar rhytides examined the onset of BoNTA-ona. Results showed that the mean time to response after treatment with a 20-U dose of BoNTA-ona was less than 2 days. In this study 48% of patients had onset on 1 day after injection and 87% of patients experienced a response by day 2. Studies analyzing the onset of BoNTA-abo indicated that the mean onset time was 3 days, with effects seen in as early as 24 hours.

BoNTB-rima has been associated with a faster onset but a shorter duration of action, with relapse times between 2 and 3 months.

Conversion ratios

Although different formulations cannot be directly compared, knowing the approximate dose conversions between BoNT products is essential to incorporate them into practice, and for studies that seek to accurately compare these products. The conversion ratio of 2.5:1.0 (BoNTA-abo:BoNTA-ona) is widely accepted in the field. In an editorial commentary on the use of the 2 formulations for cervical dystonia, Poewe stated that, based on clinical studies, the ratio of BoNTA-abo to BoNTA-ona should be no greater than 3.0:1.0. A study for the cosmetic use of BoNT used electromyographic activity to determine more precisely the dose conversion. This study enrolled 26 patients, each of whom was randomly assigned to receive BoNTA-abo and BoNTA-ona at a 3.0:1.0 ratio to each half of the frontalis muscle at 3 points of injection. Electromyographic activity was then measured at intervals as long as 20 weeks after the first injections. Results showed that at a 3.0:1.0 conversion ratio, the BoNTA-abo had a longer duration of effect. These results were interpreted as an indication that the true conversion ratio must be less than 3.0:1.0, because an accurate conversion ratio would have both formulations with equal duration of effect. Lowe and colleagues compared the duration of effect between BoNTA-abo and BoNTA-ona in the glabella using a dose ratio of 2.5:1.0. The study showed a greater duration of effect in patients treated with the 20-U dose of BoNTA-ona.

Diffusion

The toxin’s ability to remain relatively localized at the site of injection is essential for the safety of BoNT treatments. Uncontrolled spread, diffusion, or migration of the toxin not only increases the risk of distal and systemic effects but also makes the aesthetic outcome unpredictable for the treating physician.

An often-cited study by Trinidade de Almeida and colleagues compared the area of anhidrosis of each side of the forehead after injection of BoNTA-ona and BoNTA-abo to contrast their diffusion properties. Either side of each subject’s forehead was randomized to receive either BoNTA-ona or BoNTA-abo at 2 injection points (medial and lateral). The side of the forehead randomized to BoNTA-ona received 3 U at each injection point. The alternate side was then randomized to receive an equal volume of injection of BoNTA-abo but with a dose ratio of 1.0:2.5, 1.0:3.0, or 1.0:4.0. An iodine and starch test across the forehead was used to highlight the areas of anhidrosis. The halos of anhidrosis could then be measured and compared. The important factor in this study is that the injections were kept at constant volume regardless of the dose ratio. The anhidrotic halo for BoNTA-abo was found to be larger at all dose ratios from week 1 to month 6 compared with the halos for BoNTA-ona.

Studies comparing BoNTA-ona and BoNTA-inco showed that the complex proteins in BoNTA-ona do not influence the diffusion profile. BoNTB consistently showed the highest local and systemic diffusion properties. The B serotype not only produced a greater radius of toxin diffusion but also had a significantly higher number of associated side effects. Brodsky and colleagues summarize that neither molecular weight nor the presence of complexing proteins seem to affect diffusion, but properties intrinsic to the drug, accurate muscle selection, and dilution, volume, and dose injected are factors that influence diffusion.