Basic science: BOTOX® Cosmetic

4 Basic science


BOTOX® Cosmetic



Conor J. Gallagher




Summary and Key Features




• The introduction of BOTOX® Cosmetic (onabotulinumtoxinA) into aesthetic dermatology has revolutionized the management of facial lines


• Botulinum neurotoxins are biological products, synthesized by bacteria, then purified, formulated, and packaged into minute quantities for medical use


• Seven different serotypes of botulinum toxins occur in nature (types A through G), although most clinical products, including onabotulinumtoxinA, are based on the A serotype


• Botulinum toxin type A has a well-defined mechanism of action; at the neuromuscular junction, it reduces acetylcholine release from motor nerves and inhibits muscular contractions


• Clinical and preclinical data suggest that onabotulinumtoxinA may also act on nociceptive neurons


• Each commercially available botulinum toxin product is a unique biological therapeutic, with a distinct structure, formulation, unit strength and clinical profile


• As biologics, the doses of botulinum neurotoxins are expressed in units of biological activity that are not interchangeable or convertible among different products


• Recent analyses demonstrate that the onset of effect of onabotulinumtoxinA in the management of glabellar lines occurs within 24 hours and that benefits last at least 4 months


• Although all botulinum toxin products may stimulate antibody formation, the immunogenicity profile of onabotulinumtoxinA is well characterized


• The clinical efficacy and safety profile of onabotulinumtoxinA in facial lines are well understood by skilled practitioners



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).


image

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).


image

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).



Dissociation of NAPs from the 150 kDa neurotoxin


For aesthetic use, onabotulinumtoxinA is injected into muscle tissue where the neurotoxin protein complex is disassembled and the 150 kDa core neurotoxin is released to bind to its target cells. Dissociation of NAPs from the 150 kDa neurotoxin is a function of pH and ionic strength, with exposure to more basic pH and increasing ionic strength facilitating separation of the core from the surrounding accessory proteins. The process of dissociation of the complex in muscle has never been studied, and therefore the time taken for complete dissociation of the neurotoxin complex in vivo is currently unknown.


Eisele and colleagues have recently speculated that dissociation of the neurotoxin complex occurs in the vial immediately upon reconstitution in clinical practice. However, the pH of unbuffered normal saline (either unpreserved or bacteriostatic) may range between 4 and 7, with a nominal pH of 5.5. This is very close to the isoelectric point for the botulinum toxin type A complex, at which the complex is most stable; thus dissociation in the vial is highly unlikely. Indeed, the botulinum toxin type A complex is stable at this pH.



Binding

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Mar 12, 2016 | Posted by in General Surgery | Comments Off on Basic science: BOTOX® Cosmetic

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