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Basic Science of Neuromodulators in Facial Aesthetics

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Mitchell F. Brin, MD

Allergan Aesthetics, an AbbVie Company, Irvine, CA, USA
University of California, Irvine, CA, USA
Brin_Mitchell@Allergan.com
2525 Dupont Dr, Irvine, CA 92612
714-246-4429

Amy Brideau-Andersen, PhD

Allergan Aesthetics, an AbbVie Company, Irvine, CA, USA
Brideau-andersen_Amy@Allergan.com
2525 Dupont Dr, Irvine, CA 92612
714-246-4500

John Maltman, PhD

Allergan Aesthetics, an AbbVie Company, Irvine, CA, USA
John.Maltman@allergan.com
2525 Dupont Dr, Irvine, CA 92612
714-246-3165

Stephanie Manson Brown, MD, MBBS, MRCS

Allergan Aesthetics, an AbbVie Company, Marlow, UK
Brown_Stephanie@allergan.com
Marlow International, Parkway, Marlow SL7 1YL, UK
+44 1628 497207

Funding Disclosures

This work was sponsored by Allergan Aesthetics, an AbbVie Company. Writing and editorial assistance was provided to the authors by Regina Kelly, MA, of Peloton Advantage, LLC, an OPEN Health company, and funded by AbbVie. Neither honoraria nor other form of payment was made for authorship.

Author Disclosures

Amy Brideau-Andersen: is an employee of AbbVie, and may hold AbbVie stock
Mitchell F. Brin: is an employee of AbbVie, and may hold AbbVie stock
John Maltman: is an employee of AbbVie, and may hold AbbVie stock
Stephanie Manson Brown: is an employee of AbbVie, and may hold AbbVie stock

Abstract

The potent neuromodulatory effects of the toxin produced by the bacterium Clostridium botulinum were recognized over a century ago when progressive paralytic symptoms of a food-borne illness, botulism, were first documented by a medical officer around the late 18th century. The first therapeutic research use of botulinum toxin, the treatment of strabismus, occurred in 1973. In the 1980s and 1990s, the medical community serendipitously discovered the aesthetic benefits of injecting botulinum toxin into facial muscles, modulating the activity of muscles involved in wrinkle development and smoothing the skin. Today, there are multiple formulations and several known serotypes of botulinum toxin; the 2 commercially available serotypes have distinct medical indications and only type A has cosmetic. All of the botulinum neurotoxins cause temporary muscle paralysis by cleaving proteins involved in the release of neurotransmitters into the neuromuscular junction. This chapter reviews the basic science behind injectable botulinum neuromodulatory agents in facial aesthetics, with an emphasis on pharmacology and mechanism of action.

Introduction

Botulinum neurotoxins (BoNTs) are potent neuromodulators produced by the bacterium Clostridium botulinum1,2 that block vesicular neurotransmitter release and thus inhibit muscle contraction. There are 7 classical, naturally derived BoNT serotypes, identified as A through G.3-5 Serotype A (BoNT/A) has been used in therapeutic applications since the 1970s.6 In the 1980s and 1990s, the medical community serendipitously discovered the aesthetic benefits of injecting botulinum toxin into facial muscles, modulating the activity of muscles involved in wrinkle development and smoothing the skin. Today, the most well-known BoNT/A formulations are those with facial aesthetic indications, which include onabotulinumtoxinA (Botox/Botox Cosmetic/Vistabel/Vistabex), abobotulinumtoxinA (Dysport/Azzalure/Reloxin), incobotulinumtoxinA (Xeomin Cosmetic/Xeomin/Bocouture), and prabotulinumtoxinA (Jeuveau/Nabota/Evosyal/Nuceiva). Here we review the history and basic science behind injectable botulinum neuromodulatory agents in facial aesthetics, with an emphasis on pharmacology and mechanism of action.

Neuromodulator discovery and early aesthetic use

The effects of BoNT on motor and autonomic nerve function were first recognized over a century ago.7,8 Around the late 18th century, a medical officer and poet named Justinus Kerner documented progressive muscle paralysis from a lethal case of botulism, a food-borne illness with symptoms that also included mydriasis, diplopia, and gastrointestinal distress.7,8 An 1895 outbreak of botulism in Belgium resulted in the discovery of Clostridium botulinum by Emile Pierre van Ermengem, a professor of bacteriology.9 Inspired by the discovery of Daniel Drachman in 196510 that chick embryo muscles became paralyzed following BoNT injection, the ophthalmologist Dr. Alan Scott pioneered the use of neurotoxin in 1973 to treat strabismus, which was considered the first therapeutic research use of BoNT/A.11 He named the formulation “Oculinum.”12 Thereafter, in the 1980s and 1990s, small amounts of BoNT/A were used effectively in clinical research to temporarily relax facial and eye muscles to treat hemifacial spasm, strabismus, dystonia, and blepharospasm.7,13 OnabotulinumtoxinA was the first commercially available BoNT/A neuromodulator, having initially received US Food and Drug Administration approval, as Oculinum, to treat strabismus and blepharospasm in 1989.14-16 Oculinum was commercialized by Allergan and the trade name was changed to BOTOX in 1992.17

Patients undergoing treatment for blepharospasm and hemifacial spasm noticed periorbital wrinkles subsiding after injections of onabotulinumtoxinA.18 In 1987, Vancouver-based Drs. Jean and Alastair Carruthers (an ophthalmologist and a dermatologist, respectively) discovered the cosmetic benefits of BoNT/A when their patients who received routine blepharospasm injections reported the smoothing of their upper facial lines.8,18 At the same time, New York-based neurologist Dr. Mitchell Brin and otolaryngologist Dr. Andrew Blitzer discovered similar aesthetic benefits for facial rhytids, especially in the periorbital area, among patients being treated for facial spasms, including hemifacial spasm.19 In 1989, Drs. Clark (plastic surgeon) and Berris (oculoplastic surgeon) reported use of BoNT/A for aesthetic treatment,20 ie, for restoration of facial symmetry in a patient who had a face lift with rhytidectomy complicated by facial nerve damage involving the frontalis.11 Later, in Canada in 2001 and in the United States in 2002, BoNT/A, under the trade name BOTOX Cosmetic,21 became the first neurotoxin approved by regulatory agencies for the treatment of glabellar lines.16

BoNT/A PHARMACOLOGY, MECHANISM OF ACTION, AND NONINTERCHANGEABILITY

Knowledge of the fundamental steps in muscle contraction provides a basis for understanding neuromodulator mechanism of action and pharmacology. Muscles contract in response to signals from the brain passing from peripheral nerve to muscle, triggering an increase in the calcium concentration of the cytoplasm of nerve endings and allowing exocytosis of synaptic vesicles containing the neurotransmitter acetylcholine. Released acetylcholine crosses the nerve junction to activate receptors on muscle fibers, leading to stimulation of muscle contraction.22 A complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins enables the process of synaptic vesicle fusion and neurotransmitter release. The neuronal SNARE complex is composed of 3 proteins: synaptosomal-associated protein of 25 kD (SNAP-25), vesicle-associated membrane protein (VAMP, or synaptobrevin), and syntaxin.14,22,23

BoNTs are produced by clostridial bacteria as protein complexes of various sizes, with neurotoxin accessory proteins (NAPs) to protect and stabilize the core 150-kDa neurotoxin.24-28 BoNTs are di-chain molecules, consisting of a heavy (100 kD) chain and a light (50 kD) chain linked by a disulfide bond.5,29 All BoNTs are zinc-dependent metalloproteases1,30; they block the release of acetylcholine at neuromuscular junctions by cleaving SNARE proteins required for vesicle docking and fusion.24 This in turn prevents signal transduction across synaptic junctions and causes chemical denervation, resulting in temporary relaxation of the muscle.20-22 The 7 previously mentioned BoNT serotypes differ on the molecular level and are distinguished by their SNARE substrate specificity, SNARE protein cleavage position, and duration of neuromodulatory effect. BoNT/A, which cleaves the SNAP-25 protein of the SNARE complex, has the longest-lasting neuromodulatory effect and is the most commonly used commercially available subtype.4,5,22 The only other currently commercially available serotype is BoNT/B,22 which cleaves a different protein component of the SNARE complex (VAMP/synaptobrevin) and is approved in the US for the treatment of cervical dystonia and chronic sialorrhea.5,31

Key steps in the neuromodulating effects of BoNT/A on target peripheral muscles include receptor-mediated cell binding and internalization, translocation of the protease domain (light chain) into the cytosol, and proteolytic cleavage of SNAP-25 (Figure 1).30,32,33 Briefly, following injection, BoNT/A is distributed to the extracellular space where the neuromodulator comes into contact with specific receptors along nerve terminals. The heavy chain of BoNT/A binds to these receptors and is internalized via endocytosis. Once in the endosome, the light chain of BoNT/A translocates across the membrane and is released into the cytosol where it cleaves SNAP-25, thereby preventing vesicle fusion with the nerve terminal membrane and, in cholinergic neurons, blocks release of acetylcholine.33,34

Figure 1. BoNT/A Mechanism of Action
Figure 1. BoNT/A Mechanism of Action

BoNT/A, botulinum toxin A; SNAP-25, synaptosomal-associated protein, 25 kD; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor

Adapted from Burstein R, et al. Cephalalgia. 2014;34(11):853-869.35

Because BoNT products are manufactured, formulated, and tested using techniques that differ across manufacturers,25,36 their preclinical characteristics, clinical properties, and safety profiles may differ, and dosing units of the products are not interchangeable, as stated in product labeling.21,25,37 Understanding the concept of noninterchangeability of BoNT products is important to help ensure adherence to manufacturer labeling and guidance and to inform clinicians on the safe and effective use of these products.

A fundamental reason for the noninterchangeability of BoNT/A products is their biological nature: as bacterially produced proteins, they are much larger with more complex molecular structure than chemically synthesized drugs, and even small modifications to the manufacturing process can alter the protein’s structure and biological activity. In addition to differences among BoNT/A products in several steps of the manufacturing process, each manufacturer employs its own proprietary assay for testing potency units using a product-specific reference standard, meaning that units of biological activity are specific to each product.21,25,38 Several examples of unit noninterchangeability have been confirmed in studies directly comparing the activity of BoNT/A products.37,39-41       

Another key difference between products is the presence or absence of NAPs. In nature, bacteria produce BoNTs as protein complexes of various sizes (from 300 to 900 kD for type A) depending on the strain, with NAPs to stabilize and protect the 150-kD neurotoxin from degradation.24-28; Depending on differing purification processes, BoNT/A products either retain one or more of the NAPs in complexes as produced by the bacteria (onabotulinumtoxinA, abobotulinumtoxinA, prabotulinumtoxinA) or only the 150-kD neurotoxin itself (incobotulinumtoxinA).5,16,25,36,42

The translation of these manufacturing and testing differences between BoNT/A products into in vivo differences was suggested by preclinical dose-response studies of muscle weakening efficacy. In these studies, comparison of 3 BoNT/A products reported significant differences in unit potency under the experimental conditions employed.25,40

Overview Of Facial Aesthetic Approved And Potential Indications For BoNT/A

BoNT/A has clinical utility for a variety of neuronal disorders; for example, onabotulinumtoxinA is approved for the treatment of facial wrinkles (glabellar, lateral canthal, and forehead lines) and other medical uses.3 Table 1 provides an overview of BoNT/A neuromodulators presently available and licensed commercially for facial aesthetic indications in the United States and Europe.22 Also shown are any approved indications related to overactive muscle disorders and other medical conditions, where applicable.

Injections of appropriate doses of approved BoNT/A products into forehead, glabellar, and periorbital areas are intended to temporarily improve the appearance of dynamic rhytids in the forehead, glabellar, and crow’s feet regions; additionally, BoNT/A products are sometimes used “off-label” to lift and reshape the eyebrows.20,43-47 Other reported BoNT/A uses include smoothing out grooves and lines in the midface and cheeks.20,48-50 BoNT/A has also been advocated as an effective nonsurgical option for intramuscular injections to treat masseter muscle hypertrophy51 and prominent platysma bands.52

Attributes associated with optimal skin quality have been noted with BoNT/A.53 Researchers and clinicians have observed smoothing of skin overlying injected areas, which persists beyond the immediate neuromodulating effects of the neurotoxin54 and may result from local relaxation of transverse muscle cells and tissue remodeling due to muscle inactivity. In support, a statistically significant reduction in skin roughness was noted after BoNT/A treatment via quantitation of silicon replica scanning electron microscope images.54-56 It has been speculated that intradermal injection of BoNT/A preparations may modulate sebocyte lipogenesis (reducing sebum in sebocytes) and thereby minimize skin oiliness and pore size; the observed clinical effects on sebum could be due to a fibroblast growth factor receptor–binding mechanism of action, with a direct impact on sebocytes.57

Table 1. Approved US and European BoNT/A Neuromodulators With Facial Aesthetic Indications4,5,21,38,58-72

Toxin Name
 (Non-proprietary)		First Approval Year (Country)ManufacturerIndications
OnabotulinumtoxinA1989 (US)Allergan Aesthetics, an AbbVie CompanyFacial aesthetic:
Forehead lines (US, Europe)
Glabellar lines (US, Europe)
Lateral canthal lines (US, Europe)
Therapeutic:
Blepharospasm (US, Europe)
Strabismus (US)
Cervical dystonia (US, Europe)
Axillary hyperhidrosis (US, Europe)
Adult upper limb spasticity (US, Europe)
Adult lower limb spasticity (US, Europe)
Chronic migraine (US, Europe)
Neurogenic detrusor Overactivity (US, Europe)
Overactive bladder (US, Europe)
Pediatric upper limb spasticity (US)
Pediatric lower limb spasticity (US)
Pediatric dynamic equinus foot deformity (Europe)
Hemifacial spasm (Europe)
AbobotulinumtoxinA1991 (Europe)Ipsen Ltd.Facial aesthetic:
Glabellar lines (US, Europe)
Lateral canthal lines (Europe)
Therapeutic:
Adult upper limb spasticity (US, Europe)
Adult lower limb spasticity (US, Europe)
Blepharospasm (Europe)
Cervical dystonia (US, Europe)
Pediatric upper limb spasticity (US, Europe)
Pediatric lower limb spasticity (US)
Pediatric dynamic equinus foot deformity(Europe)
Hemifacial spasm (Europe)
Axillary hyperhidrosis (Europea)
IncobotulinumtoxinA2006 (Europe)Merz Pharmaceuticals, LLCFacial aesthetic:
Glabellar lines (US, Europe)
Lateral canthal lines (Europe)
Forehead lines (Europe)
Therapeutic:
Adult upper limb spasticity (US, Europe)
Blepharospasm (US, Europe)
Cervical dystonia (US, Europe)
Hemifacial spasm (Europe)
Chronic sialorrhea (US, Europe)
Pediatric upper limb spasticity,  excluding spasticity caused by cerebral palsy (US)
PrabotulinumtoxinA2019 (US, Europe)bDaewoong Pharmaceutical/Evolus, Inc.Facial aesthetic:
Glabellar lines (US, Europe)
Therapeutic:
None
a Not approved across the region; limited to specific countries.
b2013 in Korea.

 Summary

 BoNT/A inhibits the release of the neurotransmitter acetylcholine at neuromuscular junctions by cleaving the SNAP-25 protein essential to its vesicular release, causing temporary, localized chemical denervation and muscle paralysis. The medical community serendipitously discovered the aesthetic benefits of injecting botulinum toxin into facial muscles, modulating the activity of muscles involved in wrinkle development and smoothing of the skin. Today, there are multiple formulations of botulinum toxin with different cosmetic and medical indications, providing facial aesthetic applications and benefits.

Acknowledgments

Writing and editorial assistance was provided to the authors by Regina Kelly, MA, of Peloton Advantage, LLC, an OPEN Health company, and funded by AbbVie, North Chicago, IL.

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