Introduction

The introduction of botulinum toxin type A into the field of aesthetic dermatology has profoundly impacted the clinical management of undesirable facial lines. Botulinum toxins are injected into discrete facial muscles where they act locally to reduce muscle contractions that produce skin creases, either with facial animation or at rest. The pattern of injections can be tailored to individual needs and the results in glabella have been demonstrated by Carruthers and colleagues in 2004 to last an average of 4 months.

This chapter discusses the basic science of Botox^® Cosmetic, also known in the United States and other countries by its non-proprietary name onabotulinumtoxinA. Although other botulinum neurotoxin products are available in various countries worldwide, each contains a unique protein drug substance manufactured using a proprietary technology and containing a distinct formulation of excipients. The biological activity units of each product, and hence unit doses, are not interchangeable with those of other products, as per guidelines issued by regulatory agencies in all major countries throughout North America, Europe, and Asia.

In line with the non-interchangeable nature of botulinum neurotoxins, this book includes separate chapters on each of the main botulinum neurotoxin products available for aesthetic use. In the following pages, we discuss the basic properties of botulinum toxins, including their serotypes, structure, and mechanism of action. In the sections on manufacturing, formulation, immunology, and pharmacology in facial aesthetics, the focus is on onabotulinumtoxinA and it should be noted that all dosing in this chapter refers specifically to this product. (Due to the structure and format of this book, this chapter is not fully referenced; the reader is referred to the reading list at the end of this chapter for further information.)

Serotypes and structure

Botulinum toxins are biological products produced by the bacterium Clostridium botulinum. These neurotoxins have been grouped into seven serotypes based on their immunologic properties: types A, B, C₁, D, E, F, and G.

All botulinum toxins are produced by bacteria as protein complexes consisting of a core neurotoxin molecule with a molecular mass of approximately 150 kDa and one or more associated proteins. This protein contains three distinct functional domains. The binding domain is responsible for the docking of the molecule with its specific cell surface receptors, the translocation domain is critical in allowing the catalytic domain to access the neuronal cytosol, and the catalytic domain is responsible for the enzymatic activity that ultimately interferes with neurotransmitter release (Fig. 4.1).

Figure 4.1 Molecular structure of the botulinum toxin type A 150 kDa core neurotoxin. The three functional domains are color coded: yellow: light chain catalytic domain with a zinc atom shown in blue; red: heavy chain translocation domain; green: heavy chain binding domain.

Image courtesy of Lance E. Steward PhD, Allergan Inc.

Associated with the core neurotoxin are one or more additional proteins that are commonly known as accessory proteins or neurotoxin-associated proteins (NAPs). For type A botulinum toxins these consist, in part, of a number of proteins that were identified initially by their capacity for agglutination of blood, and thus are known as hemagglutinin (HA) proteins. An additional protein that is not agglutinating is always associated with the core neurotoxin and this is referred to the NTNH or non-toxic, non-hemagglutinin protein. Based on the serotype a variety of neurotoxin complex sizes may be produced by the bacterium. Type A-producing strains synthesize complexes that are of 300 kDa, 500 kDa or 900 kDa in molecular mass.

Role of NAPs

Given that botulinum toxins are always produced by the bacteria as protein complexes, this assembly may have some evolutionary advantage. NAPs serve a number of critical roles in protecting the core neurotoxin protein from harsh environmental conditions and events such as proteolytic degradation, pH stress, and thermal stress. In vitro studies by Chen and co-workers have confirmed that the complex botulinum neurotoxin shows greater resistance than the uncomplexed toxin to degradation by pepsin, gastric juices, and pancreatic enzymes.

Although the physiological environment of the digestive tract differs from that of skeletal muscle into which therapeutic botulinum neurotoxins are typically injected, the integrity of the protein complex is likely retained for at least a period of time after injection. Muscle tissue contains a plethora of extracellular enzymes that degrade proteins. The effects of muscle proteases on botulinum neurotoxins have not been investigated; however, it is possible that NAPs may transiently protect the core neurotoxin from enzymatic attack following intramuscular injection.

Limited information is available regarding the role of NAPs in the clinical use of botulinum neurotoxins. However, multiple botulinum toxin type A products are currently available for clinical use, and one of the most prominent ways in which they differ is in the amount of NAPs that they contain. Botox^® (onabotulinumtoxinA; Allergan, Inc., Irvine, California, USA) contains only the purified form of a 900 kDa complex, according to the company’s prescribing information. Another product, Xeomin^® (incobotulinumtoxin type A; Merz Pharmaceuticals, Frankfurt am Main, Germany), contains only botulinum toxin type A molecules from which the NAPs have been removed, according to the prescribing information. Hambleton reported that Dysport^® (abobotulinumtoxinA; Ipsen Biopharm Ltd., Wrexham, UK) contains botulinum toxin type A complexes of approximately 300–500 kDa. In attempting to understand how these products behave in the muscle following injection, the role of NAPs may be an important area of future study.

Manufacturing

The production of botulinum toxin protein products begins with fermentation of the clostridial bacteria in an anaerobic environment, which results in the synthesis and release of neurotoxin into the bacterial broth. In some bacterial strains the 150 kDa polypeptide is ‘nicked’, separating the protein into a 50 kDa light chain and a 100 kDa heavy chain, which remain closely associated via a disulfide linkage.

Following the fermentation step, the neurotoxin is purified from the bacterial broth. Each manufacturer has its own proprietary methodology for purification of the neurotoxin, excluding the bacterium, resulting in a unique biological product. In the purification of onabotulinumtoxinA, Allergan generally follows the methodology of Schantz, which yields a highly purified crystalline neurotoxin with a homogenous molecular mass distribution of approximately 900 kDa.

Formulation

In order to enhance the stability of botulinum toxins from the point of manufacture to the point of injection into patients, all products are formulated with excipients. For a 100 U vial of onabotulinumtoxinA, these excipients include human serum albumin (0.5 mg) and NaCl (0.9 mg). Human serum albumin acts as a stabilizer and also enhances the recovery of the neurotoxin from the glass vials. Albumin may also act as a cryoprotectant and serve to prevent protein aggregation.

Mechanism of action

Following intramuscular injection, botulinum neurotoxins inhibit the release of acetylcholine (ACh) from motor nerve terminals, resulting in reduced muscular contractions. This inhibition occurs in multiple steps referred to as binding, internalization, translocation, and cleavage. Through a similar process, botulinum neurotoxins also inhibit acetylcholine release from autonomic nerve terminals that innervate smooth muscle or glands. Further studies have found that botulinum toxin type A exerts selective effects on the nociceptive system. These actions are described in the following text, which for onabotulinumtoxinA begins with the dissociation of the 150 kDa neurotoxin from the NAPs (Fig. 4.2).

Figure 4.2 Multiple steps in the mechanism of action of botulinum toxin type A: (1) The 150 kDa core neurotoxin is internalized into the neuron via a process of endocytosis. (2) The endosome is acidified, which facilitates (3) the translocation of the light chain into the neuronal cytosol. (4) Once in the neuronal cytosol the light chain of type A botulinum toxin (blue) cleaves the membrane-associated SNAP-25 (brown), one component of the SNARE complex. Another neurotoxin serotype, type B, botulinum toxin cleaves a different protein known as synaptobrevin or vesicle-associated membrane protein (VAMP; green).

Immunogenicity

Botulinum toxins are formulated biological products that, like all foreign proteins, are capable of stimulating an immune response and antibody formation. The onabotulinumtoxinA protein complex comprises the core neurotoxin and the protective accessory proteins, as discussed previously, and Goschel and co-workers found that antibodies may form against either component. Only those antibodies that inhibit the function of the core neurotoxin (neutralizing antibodies) are of clinical relevance: if selectively targeted against the neurotoxin and present in sufficient quantities, they may lead to clinical non-response. In contrast, antibodies that may potentially form against the NAPs do not affect the biological activity of botulinum toxin type A.

The immunogenicity of onabotulinumtoxinA in the management of glabellar lines has been studied in Allergan-sponsored trials conducted for the purpose of product registration. Based on a pooled population of 718 subjects receiving a 20 U dose of onabotulinumtoxinA injected into the glabella, only 2 (0.3%) seroconverted from antibody-negative status at some time during these multi-cycle trials analyzed by Naumann et al. Both subjects who developed neutralizing antibodies retained an objective clinical response to treatment throughout the studies, and were not positive at the end of the study. In a wider analysis of onabotulinumtoxinA clinical trials by the same authors, an overall rate of seroconversion of 0.5% was observed, with more than 50% of neutralizing antibody-positive patients retaining a clinical response. Importantly, the development of neutralizing antibodies was not found to be associated with skin reactions upon injection or other immune-related adverse events in any clinical indication.

Clinical pharmacology of onabotulinumtoxinA in aesthetics

The clinical profile of onabotulinumtoxinA in aesthetic medicine has been studied intensively since the early reports of its use for glabellar lines in the 1990s, and the performance of this product is known to practitioners. OnabotulinumtoxinA has been approved for 25 clinical indications worldwide, with demonstrated safety and efficacy in large muscle indications such as upper-limb spasticity and cervical dystonia, as well as small muscle indications such as strabismus, blepharospasm, and glabellar rhytides. In addition, approvals in chronic migraine, primary axillary hyperhidrosis, and neurogenic overactive bladder demonstrate a breadth of clinical data support and use unrivalled by any other botulinum neurotoxin product.

The efficacy and safety of onabotulinumtoxinA in glabellar lines has been studied in rigorous double-blind, placebo-controlled registration trials in North America by Carruthers et al and in Asia by Harii & Kawashima and Wu et al. Responder rates in these trials were uniformly high, with more than 80% of subjects exhibiting significant clinical efficacy on dynamic glabellar lines with a 20 U dose of onabotulinumtoxinA, according to a priori responder criteria (Fig. 4.3). Based on these trials, a median duration of efficacy of 120 days or 4 months may be expected in those exhibiting a clinical response. The duration of efficacy on resting glabellar lines in these trials was found to be longer than that on dynamic rhytides, with a median duration of 131 days (Fig. 4.4).

Figure 4.3 Repeat dosing efficacy of onabotulinumtoxinA in US glabellar lines trials: physician’s assessment of glabellar line severity at maximum frown. Responder rates (percentage of patients with a rating of none or mild at follow-upon the 4-point facial wrinkles scale with photonumeric guide). Results from 258 patients receiving all three botulinum toxin treatments are illustrated.

Data from Carruthers et al. 1994 J. Clinical Research 7:1–20.

Figure 4.4 Duration of efficacy of onabotulinumtoxinA in glabellar lines: results of a pooled analysis of four onabotulinumtoxinA randomized double-blind placebo-controlled trials. Panel A shows the proportion of responders demonstrating a clinical response on dynamic glabellar lines through the course of the studies. A responder was defined as a subject with an improvement from moderate or severe glabellar lines to mild or no glabellar lines. Panel B shows the proportion of responders demonstrating a clinical response on glabellar lines at rest through the course of the studies. For this analysis a responder was defined as a subject with an improvement of 1 point on the 4-point facial wrinkle scale with photonumeric guide.

Onset of the clinical effect was not systematically studied in the registration trials for onabotulinumtoxinA. However, in a recent study by Beer and colleagues, more than 90% of physicians and subjects observed onset within 3 days of injection and almost 50% of subjects reported evidence for an onset of clinical benefit within 24 hours of injection.

The safety of onabotulinumtoxinA in the management of facial lines has been evaluated in a systematic meta-analysis by Brin et al. published in 2009. This analysis was based on nine manufacturer-sponsored clinical trials of onabotulinumtoxinA (two on lateral canthal lines and seven on glabellar lines), and included 1678 subjects who received 3–18 U per side for lateral canthal lines or 10 U or 20 U total dose for glabellar lines. In these studies, the overall incidence of adverse events was not different between the onabotulinumtoxinA- and placebo-treated groups. Eyelid ptosis (1.8%) and eyelid sensory disorder (characterized by feelings of tightness, pressure, or a heaviness to the eyelids, 2.5%) were the only adverse events that occurred at a significantly higher rate in the onabotulinumtoxinA than in the placebo group, and this was found only in the glabellar lines population. In the assessment of treatment-related adverse events, eyelid edema was also significantly more frequent in the onabotulinumtoxinA- than the placebo-treated group. As the number of treatment cycles with onabotulinumtoxinA increased, the incidence of all three of these adverse events decreased.

The dose-response effects of onabotulinumtoxinA in glabellar lines have been evaluated in randomized trials. Results of a 2005 study by Carruthers showed that in females 20–40 U of onabotulinumtoxinA produce a longer duration of action and lower relapse rate than 10 U of onabotulinumtoxinA, but indicated no differences in efficacy among the 20 U, 30 U, and 40 U doses. These findings suggest that, when averaged across a broad group of female recipients, the labeled dose of 20 U would appear to be the optimal dose for most patients.

Although anecdotal experience has suggested the accrual of cumulative benefits in patients who receive regular treatment with onabotulinumtoxinA in glabella, this has only recently been confirmed in clinical trials by Dailey and co-workers. In this study, 50 botulinum toxin-naïve subjects received treatment of glabellar lines at regular 4-monthly intervals for 20 months. During this period, their glabellar line severity never returned to baseline and in fact improved. Upon cessation of treatment (as part of the study protocol), the subjects continued to retain improvements over baseline out to the final timepoint of 6 months following their last treatment.

Approvals

The approvals for onabotulinumtoxinA in facial lines vary based on country, with a broad indication of upper facial lines (glabella, frontalis and crow’s feet) approved in some regions, glabella and crow’s feet approved in others, and only glabellar lines approved in some. Dose-ranging studies enrolling over 200 subjects with onabotulinumtoxinA in crow’s feet lines have demonstrated significant efficacy at doses ranging from 6 to 18 U per side. Additional larger-scale clinical trials for onabotulinumtoxinA in crow’s feet lines will conclude in 2012.

Unique dosing of neuromodulators

As noted previously, botulinum neurotoxins are biological products – proteins derived from bacteria. Biological products are distinguished from conventional drugs by a number of properties, including their large size, complex three-dimensional structure, and sensitivity to manufacturing methods. Regulatory agencies have determined that botulinum toxin products cannot have generic substitutes (ie, there are no biogenerics in this class), and furthermore there are no biosimilars.

As biological products with an enzymatic mechanism of action, doses of botulinum neurotoxins are measured in units of biological activity. Unlike some other classes of biologics, botulinum toxins do not have a universal reference preparation for standardizing unit potency. Typically, the biologic activity of botulinum neurotoxin products is determined in an LD50 bioassay, with each manufacturer employing a different methodology, using different diluents and a unique reference standard. For this reason, regulatory agencies are clear about the lack of comparability of units between commercially available preparations.

Although it may seem convenient to assume that either the units per vial or the labeled doses for particular indications suggest a conversion ratio between products, in fact, per regulatory agency conclusions, unit doses are not interchangeable among different botulinum neurotoxin products even if products contain the same number of labeled units per vial. This lack of unit interchangeability is demonstrated by the results of a study by Hunt & Clarke that evaluated one of the botulinum toxin type A products (incobotulinumtoxinA) in the manufacturer’s assay used to evaluate units of another product (onabotulinumtoxinA). Results showed that incobotulinumtoxinA has different biological activity when tested in the Allergan LD₅₀ assay used to define a unit of onabotulinumtoxinA than when tested in the Merz LD₅₀ assay, which defines the unique potency of incobotulinumtoxinA. These results illustrate the differences between assays, which underlies the individual potency of units of each botulinum toxin product.

Underscoring the differences in units between different botulinum neurotoxin products, regulatory agencies have determined that botulinum toxin products cannot have generic substitutes, and furthermore there are no biosimilars. The United States Food and Drug Administration (US FDA) and US Adopted Names Council (USAN) review assigned all manufacturers of products marketed in the US for therapeutic or aesthetic a non-proprietary name to emphasize non-interchangeability and reduce the potential risk for medication errors. As such, Allergan – the manufacturers of Botox^® Cosmetic – adopted the non-proprietary name onabotulinumtoxinA to refer to Botox^®. Other non-proprietary names were adopted by the manufacturers of other botulinum neurotoxin products. These names are now used extensively in clinical practice and provide an important clarifying dimension to the clinical and non-clinical literature.

Another important development related to the differences in units among botulinum neurotoxin products occurred in 2011. Historically, Allergan has employed LD₅₀ assays for batch release testing of onabotulinumtoxinA; however, in July of 2011, the US FDA announced the approval of the company’s breakthrough alternative method of testing units, specific to onabotulinumtoxinA. This method uses a cell line rather than animals for routine batch release assays. As this assay gains regulatory approval worldwide, it is anticipated that it will significantly reduce the need for animal testing related to onabotulinumtoxinA. The assay is highly sensitive and extensively cross-validated against the LD₅₀ assay.

Conclusion

OnabotulinumtoxinA is well established as a valuable management strategy for upper facial lines with varying facial regions approved in different countries. This biological product is based on the A serotype of botulinum toxin, derived from the Clostridium botulinum bacteria. The 900 kDa neurotoxin protein complex is isolated and purified from the bacteria and formulated into an injectable therapy using state-of-the-art manufacturing methods.

Following injection into muscles, botulinum toxin type A inhibits the release of acetylcholine at the neuromuscular junction, thereby reducing muscular contractions. Beneficial effects of onabotulinumtoxinA on glabellar lines are detectable within 24 hours and last a median of 4 months. The safety of onabotulinumtoxinA in the treatment of glabellar lines has been established in individual clinical trials, as well as a meta-analysis in which the only adverse events that occurred at a significantly higher rate than with placebo were local effects in the eyelid region. Dose-ranging studies have found that 20 U of onabotulinumtoxinA is the optimal dose for most patients when treating glabellar lines. This dose applies only to onabotulinumtoxinA, as units are not interchangeable among botulinum neurotoxin products.

The clinical efficacy and safety profile of onabotulinumtoxinA in facial lines is well understood by skilled practitioners. Allergan is pursuing clinical studies evaluating additional uses of onabotulinumtoxinA as part of our continued exploration into the properties of this biological product that will likely be a useful tool for aesthetic dermatologists and their patients for years to come.

Acknowledgments

The editorial assistance of Mary Ann Chapman, PhD is gratefully acknowledged.