The Academy for the

Advancement of Aesthetics

Module: Botulinum Toxin Type A

Course A: The Science Behind Botulinum Toxin Type A

Introduction:
The Science Behind BoNT/A
► Welcome to Course A: The Science Behind Botulinum Toxin Type A.
► This on-demand course will describe the history of BoNT/A, the products currently available for
use in aesthetic medicine, and their molecular structure and MOA.
► The pharmacological similarities and differences between the BoNT/A products will be
summarised, as well as the key studies documenting their use.

► Learning objectives
Upon proper completion of this activity, participants should be better able to:
► Compare the storage requirements and stability after opening for various BoNT/A products
► Describe the mechanism of action of botulinum toxin type A (BoNT/A) in facial aesthetic medicine
► Differentiate BoNT/As for facial aesthetic medicine based on indications, efficacy, and safety

MOA, mechanism of action 2

The History of Botulinum Toxin
► The existence of botulinum toxin (BoNT) was first investigated in
the early 1800s, after a food poisoning outbreak from undercooked
sausages. BoNT has been studied extensively since, in both
therapeutic and aesthetic uses.
► BoNTs are potent neurotoxins naturally produced by the
anaerobic bacterium Clostridium botulinum. Different strains
of the bacterium produce distinct toxins: serotypes A to G.
The A, B, and E serotypes cause human botulism, with
the activities of types A and B enduring longest in vivo,
from several weeks to months.1
► Clostridium botulinum. A species of anaerobic,
Gram-positive, rod-shaped bacteria that produce BoNTs.
Li B et al. Molecules. 2011;16:202-220.
Image top, © RoyaltyStockPhoto | Dreamstime.com.
Image bottom from the US Centers for Disease Control and Prevention's Public Health Image Library, ID #2107. 3
The History of Botulinum Toxin (continued)
► There are 7 BoNT serotypes: A, B, C1, D, E, F, and G.
► Each serotype is produced by different strains of C botulinum.
► These bacterial strains are physiologically and phylogenetically distinct.
► A number of strains will produce BoNT/A; however, it is the high-yielding
Hall strain of C botulinum that produces the most potent form of BoNT/A.1,2
► BoNT/As are the most common form of BoNT used in aesthetic clinical practice.3

1. Ng V et al. Genomics. 2014;103:94-106. 2. Ting P, Freiman A. Clin Med. 2004;4:258-261. 3. Carruthers J, Carruthers A.
www.uptodate.com/contents/overview-of-botulinum-toxin-for-cosmetic-indications. Accessed 1/5/17. 4
The History of Botulinum Toxin:
Key Milestones
1817-1822
First description of symptoms of food-borne botulism
The first accurate and complete descriptions of the symptoms of food-borne botulism were published
by German poet and district medical officer Justinus Kerner. While he did not defining the suspected
"sausage poison" or "fatty poison," he developed the idea of its possible therapeutic use.
1895
Clostridium botulinum first identified
Belgian scientist Émile Pierre van Ermengem identified C botulinum as the bacterium causing
botulism, a condition that causes paralysis of motor muscles and ptosis.
1928
BoNT/A first isolated
BoNT/A was first isolated by Dr Herman Sommer at the University of California.

1946
BoNT/A purified
Dr. Carl Lamanna and colleagues purified BoNT/A in crystalline form.
Carruthers A et al. Dermatol Surg. 2013;39:493-509. 5
The History of Botulinum Toxin:
Key Milestones1 (continued)
1950s
BoNT/A MOA elucidated
Dr Vernon Brooks found that BoNT/A inhibits release of the neurotransmitter, acetylcholine, from motor
nerve endings when injected into muscle, which subsequently reduces the target muscle’s activity.
1973
BoNT/A used for treating strabismus in animal studies
Dr Alan Scott, an eye surgeon based in California, studied the efficacy of BoNT/A as a possible
treatment for strabismus – his experiments in monkeys proved successful.2
1946
BoNT/A purified
Dr. Carl Lamanna and colleagues purified BoNT/A in crystalline form.
1980s
BoNT/A first used in humans
The first clinical trial to establish the efficacy and tolerability of BoNT/A (comprising more than
7000 patients with strabismus) was published.
1. Carruthers A et al. Dermatol Surg. 2013;39:493-509. 2. Scott A et al. Invest Ophthalmol. 1973;12:924-927. 6
The History of Botulinum Toxin:
Key Milestones1 (continued)
1989
First BoNT/A product approved
Under the trade name Oculinum, the US Food and Drug Administration (FDA) approved the
first BoNT/A product for treating strabismus, blepharospasm, and hemifacial spasm in adults.

1992
First study of BoNT/A for treatment of glabellar frown lines published
During Dr Scott’s studies, an unexpected side effect was noted: an improvement in
wrinkles in the glabella. This was the catalyst for studying the use of BoNT/A in
aesthetic medicine; subsequently, the first study evaluating the efficacy of BoNT/A
in treatment of glabellar frown lines was published by Jean and Alastair Carruthers.2

2001
First approval of BoNT/A for treatment of glabellar frown lines
In 2001, onabotulinumtoxinA was approved in Canada and the United Kingdom for
the treatment of glabellar lines. This was followed by US FDA approval in 2002.
Subsequently, additional BoNT/A products (abobotulinumtoxinA and
incobotulinumtoxinA) were approved for this and other indications.
1. Carruthers A et al. Dermatol Surg. 2013;39:493-509. 2. Carruthers J, Carruthers A. J Dermatol Surg Oncol. 1992;18:17-21. 7
BoNT/A Products: Introduction
► BoNT/A is now considered one of the most widely researched agents in
the world.1
► BoNT/A products are injectable treatments with a number of aesthetic
indications.
► Commercially available BoNT/A products are not interchangeable and
differ in their manufacturing process, formulations, and dosing.2-5
► It is important to understand the differences between the various
preparations
to ensure proper application and avoid errors.

1. Carruthers A et al. Dermatol Surg. 2013;39:493-509. 2. Bocouture. Summary of Product Characteristics. Merz, 2015. 3. Azzalure. Summary of Product
Characteristics. Galderma, 2015. 4. Vistabel. Summary of Product Characteristics. Allergan, 2015. 5. Brin MF. Muscle Nerve Suppl. 1997;6:S208-220. 8
Summary of US FDA-Approved BoNT/A
Formulations and Manufacturing Characteristics
AbobotulinumtoxinA IncobotulinumtoxinA OnabotulinumtoxinA
Manufacturer Ipsen Ltd Merz Pharmaceuticals GmbH Allergan Inc.
Packaging, units per vial Varies by country Varies by country Varies by country
Freeze-dried lyophilised Freeze-dried lyophilised Vacuum-dried powder
Presentation
powder for reconstitution powder for reconstitution for reconstitution
Reconstitution medium 0.9% NaCl (sterile solution) 0.9% NaCl (sterile solution) 0.9% NaCl (sterile solution)
Excipients Lactose, HSA Sucrose, HSA NaCl, HSA
Complexing proteins Yes No Yes
pH 5–7 5–7 5–7
2–8 ≤25 2–8
Storage before reconstitution, ⁰C
(refrigeration) (room temperature) (refrigeration)
Glabellar lines (US)
Upper facial lines (EU), includes Glabellar lines,
Indication(s) Glabellar lines
glabellar lines, lateral periorbital lateral canthal lines
lines, horizontal forehead lines
Durability, mo 24 36 36
Dysport: within 8 h
Use after opening Within 24 h, store at 2⁰–8⁰C Within 24 h, store at 2⁰–8⁰C
Azzalure: within 4 h, store at 2⁰–8⁰C

PLEASE NOTE: PRODUCT AVAILABILITY AND INDICATION VARIES BY COUNTRY.

PLEASE REFER TO YOUR LOCAL SUMMARY OF PRODUCT CHARACTERISTICS OR PRESCRIBING INFORMATION.
HSA, human serum albumin 9
BoNT/A Products: Key Practical Differences
► Two key practical differences between the BoNT/A products are their
storage requirements and stability after opening.
► It is important that products are stored at the correct temperature as,
prior to reconstitution, incobotulinumtoxinA can be stored at room
temperature (≤25⁰C) while both abobotulinumtoxinA and
onabotulinumtoxinA need to be refrigerated.
► For this reason, abobotulinumtoxinA and onabotulinumtoxinA must be
shipped using a cold supply chain.

10
BoNT/A Products: Characteristics
► Once reconstituted, all botulinum products should be stored in the
refrigerator, and must be used within 4–24 hours after opening
depending on product.
► All commercially available BoNT/A products contain the protein,
albumin, an excipient that is added to stabilize the product and to aid
in the recovery of the neurotoxin from the vial.
► The albumin is human serum albumin (HSA) and is reported to be
generally nonimmunogenic (ie, does not cause an immune response).

Naumann M et al. J Neural Trans. 2013;120:275-290. 11

BoNT/A: Molecular Structure
► All BoNT/A products contain a 150-kilodalton (kDa) neurotoxin.
This neurotoxin is responsible for the clinical effects of BoNT/A.
► The neurotoxin is synthesized as a relatively inactive single-chain
polypeptide.
► The polypeptide is activated when it is proteolytically cleaved into a
100-kDa heavy chain and a 50-kDa light chain, connected by a
disulfide bridge.1
► The heavy chain promotes translocation of the light chain across
cellular membranes once injected into the body.2

1. Dressler D et al. Arq Neuropsiquiatr. 2005;63:180-185. 2. Frevert J. Drugs R D. 2015;15:1-9. 12

Structure of the Botulinum Neurotoxin
► The heavy chain is responsible for
binding toxin to presynaptic
receptors of the nerves and
promotes translocation of the light
chain across the endosomal
membrane
► The botulinum neurotoxin (150 kDa)
is associated with nontoxic proteins
to form a large molecular weight
complex (300–900 kDa).
► These nontoxic proteins are called
complexing proteins.

Reprinted from Laskowski RA et al. FEBS Letters. 2009;583:1692-1698. With permission from Elsevier. 13
BoNT/A: Molecular Structure (continued)

► While all BoNT/A products contain the neurotoxin, each product differs in the
presence and amount of complexing proteins.

14
BoNT/A: Complexing Proteins
► A varying number of complexing proteins are noncovalently bound
to the 150-kilodalton (kDa) neurotoxin in each BoNT/A product.
► This results in the products having different molecular weights and
3-dimensional structures.
► The complexing proteins include hemagglutinins or
nonhemagglutinin clostridial proteins.1
► The complex composition of BoNT/A products is specific to the
method of growth and purification used in production.1

1. Frevert J. Drugs R D. 2015;15:1-9. 15

BoNT/A: Complexing Proteins (continued)
A key difference between the 3 BoNT/A products is the complexing proteins they contain.1,2

Molecular mass, Complexing Total protein content, Neurotoxin

kDa Proteins ng per 100 U protein load
AbobotulinumtoxinA 500-900 Yes 4.87 0.65
IncobotulinumtoxinA 150 No 0.44 0.44
OnabotulinumtoxinA 900 Yes 5.0 0.73

1. Frevert J. Drugs R D. 2015;15:1-9. 2. Panjwani N et al. Botulinum J. 2008;1:153-166. 16

BoNT/A: Complexing Proteins (continued)
► AbobotulinumtoxinA has a molecular weight ranging from 500 to 900 kDa.2
► OnabotulinumtoxinA has a molecular weight of approximately 900 kDa.1
► Both products contain various complexing proteins.1,2

► IncobotulinumtoxinA has a lower molecular weight of 150 kDa.

► This is because the final product does not contain complexing proteins as these are
removed during the manufacturing process. Therefore, the molecular mass of
incobotulinumtoxinA is accounted for by the neurotoxin alone.

1. Dressler D et al. Arq Neuropsiquiatr. 2005;63:180185. 2. Carruthers A et al. Dermatol Surg. 2013;39:493-509. 17
BoNT/A: Complexing Proteins (continued)
► Thecomplexing proteins of the BoNT/A
complex can be visualised by Western
blotting, an analytical technique used
to detect specific proteins in a sample.
► Gel electrophoresis is used to separate
proteins by the length of the polypeptide
and the proteins are then transferred to a
membrane where they are stained with
antibodies specific to BoNT/A.

18
BoNT/A: Complexing Proteins (continued)
► During the manufacturing process for incobotulinumtoxinA, the complexing
proteins are removed via a series of purification steps.
► This yields the active neurotoxin, free from complexing proteins.
► Hence all the protein content of incobotulinumtoxin A can be attributed to neurotoxin.1

1. Frevert J. Drugs R D. 2015;15:1-9. 19

BoNT/A: The Role of Complexing Proteins
► Complexing proteins are encoded in 2 gene clusters located on the
C botulinum chromosome.
► The first cluster encodes the neurotoxin, along with nontoxin
nonhemagglutinin protein, while the second cluster encodes
3 hemagglutinin proteins.1

► It has been proposed that complexing proteins may protect

the neurotoxin when in the gastrointestinal (GI) tract, as
ingested botulinum toxin must cross the epithelium of the GI
lining before causing an effect on muscles.1

1. Frevert J, Dressler D. Biologics. 2010;4:325-332. 20

The Role of Complexing Proteins (continued)
► However, in the aesthetic setting, BoNT/A is delivered transdermally.
► Therefore, transportation across the GI tract is not relevant to clinical
efficacy.
► In addition, at physiological pH levels, the neurotoxin has been
shown to dissociate rapidly (within 1 minute) from the complex,
making it unlikely that complexing proteins are essential for stability.1

1. Brin MF. Muscle Nerve Suppl. 1997;6:S208-220. 21

The Role of Complexing Proteins (continued)

Rapid dissociation of neurotoxin and complexing proteins at physiological pH

22
The Role of Complexing Proteins (continued)
► It has also been demonstrated that there is no difference in the diffusion of the
free or complexed forms of BoNT/A following injection into the muscle.1
► Therefore, the role of complexing proteins remains unclear, but we do know that
they do not influence the storage or diffusion of the botulinum toxin products.
► However, the presence of complexing proteins in some BoNT/A products may lead to an
increased risk of developing neutralizing antibodies, which can cause suboptimal therapeutic
outcomes.
► To learn more about the possible roles of complexing proteins in BoNT/A
products refer to: Frevert J. Drugs R D. 2015;15:1-9.

1. Tang-Liu DD et al. Toxicon. 2003;42:461-469. 23

Immunogenicity
► As BoNT/A products contain foreign protein antigens, they have the potential to
induce antibody production.
► Antibodies can be raised to the neurotoxin or complexing proteins.
► Those raised to the neurotoxin that inhibit its action are termed neutralizing antibodies, and
these can lead to clinical nonresponse (defined as a lack of patient or physician satisfaction
with the therapeutic outcome).
► Although aesthetic indications require lower doses of BoNT/A than therapeutic
indications,1 all treatments require repeated injections over time, which also
increases the risk of antibody formation.2

1. Kerscher M et al. J Drugs Dermatol. 2013;12:e111-120. 2. Benecke R. Biodrugs. 2012;26:e1–e9. 24

Neutralizing Antibodies
Inhibit the Action of BoNT/A
► The immunogenicity of abobotulinumtoxinA,
incobotulinumtoxinA, and onabotulinumtoxinA
has been evaluated in New Zealand white
rabbits.
► Data showed that, after repeated intradermal
injection, incobotulinumtoxinA was the only
preparation that did not induce the formation
of neutralizing antibodies.1
► Furthermore, in a study of 42 patients who
Antibody
received BoNT/A (onabotulinumtoxinA or
Antibody
abobotulinumtoxinA) for spasticity for at least
2 years, neutralizing antibodies were detected
in 5 patients, 3 of whom were nonresponders
to BoNT/A.2
► Therefore, reducing the antigenicity of a
BoNT/A preparation is desirable.

1. Blümel J et al. Neurotox Res. 2006;9:238. 2. Müller K et al. J Neural Trans. 2009;116:579-585. 25
Secondary Treatment Failure
► When a previously effective treatment no longer produces clinical benefit during
the course of therapy, this is termed secondary nonresponse.1
► Neutralizing antibodies are an important cause of secondary nonresponse.1
► The presence of complexing proteins in some BoNT/A preparations represents an additional
foreign protein load, which may heighten the potential risk for formation of neutralizing
antibodies.1,2
► Choosing a neurotoxin that is free from complexing proteins might provide a
lower risk of eliciting an immunogenic response.2
► Other excipients could promote an innate immune response.
► One possible factor could be flagellin, which was identified as a protein component of the
abobotulinumtoxinA bulk toxin.
► Flagellin is a constituent protein of the bacterial locomotor apparatus that interacts with the
Toll-Like Receptor 5, initiating an innate immune response.3

1. Benecke R. Biodrugs. 2012;26:e1-9. 2. Frevert J, Dressler D. Biologics. 2010;4:325-332. 3. Naumann M et al. J Neural Trans. 2013;120:275-290. 26
Secondary Treatment Failure (continued)
► Unfortunately, secondary treatment failure is being reported more
frequently, typically after treatment with abobotulinumtoxinA,
onabotulinumtoxinA, or both.1
► Published case reports highlight the increased risk of treatment failure
with repeated neurotoxin injections at multiple sites.2-4
► To read about 5 cases of secondary treatment failure due to neutralizing antibody
formation after the administration of conventional BoNT/A containing complexing
proteins, refer to: Torres S et al. Clin Cosmet Investig Dermatol. 2014;7:11-17.2
► To read about a case where the development of neutralizing antibodies over 6 years
of BoNT/A treatment for glabellar frown lines resulted in secondary treatment failure,
refer to: Stengel G, Bee EK. Clin Interv Aging. 2011;6:281-284.4

1. Carruthers A et al. Dermatol Surg. 2013;39:493-509. 2. Torres S et al. Clin Cosmet Investig Dermatol. 2014;7:11-17.
3. Borodic G. Ophthal Plast Reconstr Surg. 2006;22:239-240. 4. Stengel G, Bee E. Clin Interv Aging. 2011;6:281-284. 27
MOA: How BoNT/A Exerts Its Effects
After Injection Into the Human Body
► In normal nerve cells, when an action
potential depolarizes the axon terminal,
it triggers the release of neurotransmitter
from the presynaptic nerve cell into the
synaptic cleft.
► Acetylcholine is the principal neurotransmitter at
the neuromuscular junction and is stored in
membrane-enclosed synaptic vesicles.
► The soluble N-ethylmaleimide-sensitive
factor attachment protein receptor (SNARE)
protein complex, which consists of proteins
including synaptosomal-associated protein 25 (SNAP-25) and syntaxin, is found on the
membranes of synaptic vesicles and target membranes.
► The SNARE protein complex is important for docking the synaptic vesicle to the target
membrane for neurotransmitter release into the synaptic cleft.
Figure from Arnon SS et al. JAMA. 2001;285:1059-1070. Copyright © 2001 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society. 28
MOA: Key Steps of Neurotransmitter Release
in Normal Nerve Cells1,2
► Action potential depolarizes the axon terminal.
► The SNARE protein complex on the membranes of synaptic vesicles
and the target membrane dock the vesicle to the target membrane.
► Neurotransmitter stored in the vesicles is released into the synaptic
cleft via exocytosis.
► Once in the synaptic cleft, the neurotransmitter binds postsynaptic
receptors to elicit an effect on the postsynaptic cell.

1. Dressler D et al. Arq Neuropsiquiatr. 2005;63:180-185. 2. Alberts B et al. Molecular Biology of the Cell. 5th Ed. New York, NY; Garland Science: 2012. 29
MOA: How BoNT/A Interrupts the Normal
Process of Neurotransmission
How BoNT/A causes neuromuscular paralysis:
1. BoNT/A travels to the neuromuscular junction
2. BoNT/A is internalised via endocytosis
3. The light chain of BoNT/A binds to the soluble
N-ethylmaleimide-sensitive factor attachment
protein receptor (SNARE) protein complex and
proteolytically cleaves synaptosomal-associated
protein 25 (SNAP-25)
4. Docking of the synaptic vesicle to the target
membrane is inhibited because the SNARE complex cannot form
5. Acetylcholine release into the postsynaptic cleft is inhibited, resulting in neuromuscular
paralysis

These steps are illustrated in the following MOA video.

Figure from Arnon SS et al. JAMA. 2001;285:1059-1070. Copyright © 2001 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society 30
BoNT/A: MOA Video

After Viewing the Video Click the X at

the Top Right of the Video to Continue…

31
Why is Muscle Paralysis Useful?
► BoNT/A induces weakness of striated muscles by inhibiting transmission of alpha
motor neurones at the neuromuscular junction.
► This has led to its use in conditions with muscular overactivity.

Conditions That Can be Treated

with BoNT/A Injections
Strabismus
Bruxism
Chronic migraine
Blepharospasm
Cervical dystonia
Severe primary axillary hyperhidrosis
Upper limb spasticity
Overactive bladder
32
Why is Muscle Paralysis Useful? (continued)
► Similarly,in areas where muscle activity has caused the development
of dynamic lines and wrinkles, BoNT/A can be used to soften these
lines.
► This leads us to the aesthetic indications of BoNT/A.

► Please keep in mind that not all of the indications are approved so far.

PLEASE NOTE: PRODUCT AVAILABILITY AND INDICATION VARY BY

COUNTRY. PLEASE REFER TO YOUR LOCAL SUMMARY OF PRODUCT
CHARACTERISTICS OR PRESCRIBING INFORMATION.

33
Aesthetic Indications for BoNT/A
► Since their approval in aesthetics, BoNT/A injections have become
one of the most frequently requested aesthetic procedures around
the world1
► As mentioned earlier, there are now 3 BoNT/A products widely
available for aesthetic indications:
► AbobotulinumtoxinA
► IncobotulinumtoxinA
► OnabotulinumtoxinA

1. International Society of Aesthetic Plastic Surgery. ISAPS Global Statistics. www.isaps.org/news/isaps-global-statistics. Accessed: 1/6/17. 34
 Aesthetic Indications for BoNT/A (continued)
► The table below provides an overview of the aesthetic indications for the
3 most widely available BoNT/A products.1-3
Nonproprietary Name Aesthetic Indication(s)
AbobotulinumtoxinA1 • Temporary improvement in the appearance of moderate-to-severe vertical lines between the eyebrows
seen at frown (glabellar frown lines) in adults <65 years of age
IncobotulinumtoxinA2 • Temporary improvement in the appearance of moderate-to-severe vertical lines between the eyebrows
seen at frown (glabellar frown lines)
• Lateral periorbital lines seen at maximum smile
OnabotulinumtoxinA3 • Moderate-to-severe vertical lines between the eyebrows seen at maximum frown (glabellar frown lines)
• Moderate-to-severe lateral canthal lines seen at maximum smile
• Simultaneous treatment of moderate-to-severe lateral canthal lines seen at maximum smile and
glabellar frown lines seen at maximum frown

PLEASE NOTE: PRODUCT AVAILABILITY AND INDICATION VARY BY COUNTRY. PLEASE REFER TO
YOUR LOCAL SUMMARY OF PRODUCT CHARACTERISTICS OR PRESCRIBING INFORMATION.

1. Vistabel. Summary of Product Characteristics. Allergan, 2015. 2. Bocouture. Summary of Product Characteristics. Merz, 2015.
3. Azzalure. Summary of Product Characteristics. Galderma, 2015. 35
Potency of BoNT/A Products
► Potency is a measure of drug activity; more specifically, it is the amount needed to
elicit a particular effect. BoNT/A products are extremely potent agents.
► The biological effect of incobotulinumtoxinA and abobotulinumtoxinA
is determined using the mouse LD50 test, or the median lethal
intraperitoneal doses of the agents in mice.1
► Potency is therefore expressed in mouse units. The potency of
abobotulinumtoxinA is measured in Speywood units. One Speywood
unit is defined as the median lethal peritoneal dose in mice (LD50).2
► The test is conducted differently by BoNT/A manufacturers, and each manufacturer
uses a unique, product-specific reference standard for testing biological activity.

1. Brin MF. Muscle Nerve Suppl. 1997;6:S208-220. 2. Azzalure. Summary of Product Characteristics. Galderma, 2015. 36
Potency of BoNT/A Products (continued)
► Potency of onabotulinumtoxinA is measured using a cell-based potency assay
using differentiated human neuroblastoma SiMa cells, which has shown similar
sensitivity to the mouse assay.1
► Differences in the format and methodology of the potency tests applied by the
respective manufacturers prevent direct comparisons of biological activity.2
► Therefore, units of biological activity are specific to each BoNT/A product and
unit doses are not interchangeable.
► However, there have been some comparisons of biological activity of BoNT/A
products in clinical studies.
► Two studies have shown incobotulinumtoxinA to be noninferior to onabotulinumtoxinA when
a clinical conversion ratio of 1:1 (units) was used in therapeutic indications.3,4

1. Fernandez-Salas E et al. PLoS ONE. 2012;7:e49516. 2. Panjwani N et al. Botulinum J. 2008;1:153-166. 3. Benecke R et al. Neurology. 2005;64:1949-1951.
4. Roggenkamper P et al. J Neural Transm. 2006;113:303-312. 37
Potency of BoNT/A Products (continued)
► In a European, multicenter, prospective, randomized, double-blind head-to-head study
involving a total of 381 patients, incobotulinumtoxinA and onabotulinumtoxinA
demonstrated comparable response rates in the treatment of glabellar frown lines.
► Using a clinical dose conversion ratio of 1:1 (units), noninferiority of incobotulinumtoxinA to
onabotulinumtoxinA was confirmed at 1 and 3 months whether assessed by independent raters or
treating physicians.1
► A US, multicenter, prospective, randomized, double-blind head-to-head study involving
a total of 250 patients was designed and powered to test equivalence between
incobotulinumtoxinA and onabotulinumtoxinA in the treatment of glabellar frown lines
using a clinical dose conversion ratio of 1:1 (units).
► The study confirmed comparable performance of incobotulinumtoxinA and onabotulinumtoxinA in the
treatment of glabellar frown lines at 1 month and similar efficacy profiles were demonstrated at 1, 2, 3,
and 4 months, whether assessed by independent raters or treating physicians.2

1. Sattler G. Dermatol Surg. 2010;36 Suppl 4:2146-2154. 2. Personal communication, Michael Gold, MD. 38
Potency of BoNT/A Products (continued)
►A clinical conversion ratio of 1:1 (units) is typically used for
incobotulinumtoxinA and onabotulinumtoxinA.
► For abobotulinumtoxinA, the standard dose conversion ratio is
2.5 to 3 times that for incobotulinumtoxinA/onabotulinumtoxinA.1

► You can read more about the potency of BoNT/A formulations here:
Jandhyala R. J Drugs Dermatol. 2012;11:731-736.

1. Carruthers A et al. Dermatol Surg. 2013;39:493-509. 39

BoNT/A: Key Efficacy Studies for Glabellar
Frown Lines
► BoNT/A has been studied extensively in large, Phase 3, randomized
studies, demonstrating its efficacy and tolerability in the treatment
of glabellar frown lines and periorbital wrinkles.
► Next, we will present the key publications supporting the efficacy of
the different BoNT/A formulations in treating glabellar frown lines.

40
Key Efficacy Studies for Glabellar Frown
Lines: AbobotulinumtoxinA
The US aesthetic clinical trial program for abobotulinumtoxinA included 3 randomized, placebo-
controlled, double-blind studies1-3 (below) and 2 open-label, repeat-dose studies.4-6

Study Design Conclusions

Brandt F et al. • Phase 3, prospective, randomized, double- • A single treatment with abobotulinumtoxinA was
Dermatol Surg. blind, placebo-controlled, multicenter, parallel- significantly superior to placebo in the correction of
2009;35(12): group study moderate to severe glabellar lines, with comparable
1893-1901.1 • Randomized adult patients with moderate-to- tolerability.
severe glabellar lines at maximum frown
Rubin M et al. J • Phase 3, multicenter, randomized, placebo- • Multiple treatment cycles of BoNT-A were well tolerated
Drugs Dermatol. controlled, double-blind study in the majority of patients
2009;8(5):439- • Randomized adult patients with moderate-to- • There was no tachyphylaxis seen during study duration of
444.2 severe glabellar lines at maximum frown up to 23 months.
Kane M et al. • Phase 3, randomized, double-blind, placebo- • A single treatment with abobotulinumtoxinA, with the
Plast Reconstr controlled study dose based on gender and muscle mass, is well tolerated
Surg. 2009; • Randomized adult patients with moderate-to- and provides a greater and longer-lasting improvement in
124(5):1619- severe glabellar lines at maximum frown glabellar lines compared with placebo.
1629.3

4. Monheit G, Cohen J. J Am Acad Dermatol. 2009;61:421-425. 5. Cohen JL et al. Aesthet Surg J. 2009;29:S43-S49. 6. Moy R et al. Arch Facial Plast Surg. 2009;11:77-83. 41
Key Efficacy Studies for Glabellar Frown
Lines: IncobotulinumtoxinA
The key studies for incobotulinumtoxinA are shown below, including the 2 randomized, multicenter,
placebo-controlled studies that were part of the US aesthetic clinical trial program.1

Study Design Conclusions

Jones D et al. • Data pooled from 2 large, Phase 3, placebo- • Superiority of incobotulinumtoxinA over placebo for
Dermatol Surg. controlled studies treating glabellar frown lines was confirmed.
2014;40(7):776- • Post-hoc analysis of investigator-assessed and • IncobotulinumtoxinA achieved maximum responder rate
785.1 subject-assessed responder rates, mean score, of 93.1% and a long duration of treatment effect: 45.7% of
and mean change from the baseline glabellar subjects showed efficacy at 120 days.
frown line severity score
Hanke C et al. • Phase 3, prospective, randomized, double- • A single dose of 20 U of incobotulinumtoxinA is superior
Dermatol Surg. blind, placebo-controlled study to placebo in the treatment of glabellar frown lines at Day
2013;39(6):891- • Randomized adult patients with moderate-to- 30 and is well-tolerated.
899. severe glabellar lines at maximum frown
Carruthers A et • Phase 3, prospective, double-blind, placebo- • A single dose of 20 U of incobotulinumtoxinA
al. Dermatol controlled, multicenter study demonstrated efficacy and safety in the treatment of
Surg. 2013;39(4): • Randomized adult patients with moderate-to- glabellar frown lines using new FDA efficacy variables.
551-558. severe glabellar lines at maximum frown

42
Key Efficacy Studies for Glabellar Frown
Lines: IncobotulinumtoxinA (continued)
Study Design Conclusions
Sattler G et al. • Phase 3, prospective, multicenter, randomized, • IncobotulinumtoxinA is equally effective as
Dermatol Surg. rater-and patient-blind, parallel-group study onabotulinumtoxinA in treatment of glabellar frown lines.
2010;36(suppl 4): • Randomized adult patients with moderate-to- • Both preparations were well tolerated.
2146-2154. severe glabellar lines at maximum frown
Kane M, Gold M, • Phase 4, prospective, multicenter, randomized, • Study met primary efficacy endpoint, defined as ≥1-point
et al. rater-and patient-blind, parallel-group study of improvement from baseline on the Facial Wrinkle Scale at
Unpublished incobotulinumtoxinA vs onabotulinumtoxinA maximum frown, 1 month after single treatment.
data.1 • Moderate-to-severe glabellar frown lines on a • Similar efficacy profiles for the 2 treatment groups at all
4-point Facial Wrinkle Scale at maximum frown time points (1, 2, 3, and 4 months posttreatment).
Prager T et al. • Retrospective analysis of study of 1256 patients • No statistically significant differences with respect to
Clin Cosmet in Germany receiving 2 or 3 consecutive physician and subject satisfaction, dosages, and AEs
Invest Dermatol. treatments treated with BoNT/A within a 12- between incobotulinumtoxinA and onabotulinumtoxinA.
2012;5:53-58. month period in previous 2 years • In daily practice, incobotulinumtoxinA and
onabotulinumtoxinA are used at a 1:1 dose ratio and
display comparable efficacy and safety.

1. Personal communication, Michael Gold, MD. 43

Key Efficacy Studies for Glabellar Frown
Lines: OnabotulinumtoxinA
The US aesthetic clinical trial program for onabotulinumtoxinA in glabellar frown lines included a
placebo-controlled phase1,2 followed by an open-label repeat injection phase.3

Study Design Conclusions

Carruthers J et al. • Prospective, multicenter, double-blind, • Reduction in glabellar line severity was significantly
J Am Acad randomized, placebo-controlled study greater with onabotulinumtoxinA than with placebo.
Dermatol. 2002; • Adult patients with moderate-to-severe glabellar • Effect maintained for many patients through day 120.
46:840-849.1 lines at maximum frown • OnabotulinumtoxinA injections are safe and effective in
reducing the severity of glabellar lines.
Carruthers J et al. • Multicenter, double-blind, randomized, placebo- • Responder rate was significantly greater than that for
Plast Reconstr controlled, parallel-group study placebo at every follow-up visit.
Surg. 2003;112: • Randomized adult patients with moderate-to- • OnabotulinumtoxinA was remarkably safe and effective in
1089-1098.2 severe glabellar lines at maximum frown reducing glabellar lines.
Carruthers A et • Two identical, 4-month, randomized, double- • When patients received 2 additional fixed-dose
al. J Clin Res. blind, placebo-controlled trials with 8-month treatments (at 0 months and 4 months; cycles 1 and 2,
2004;7:1-20.3 open-label extension respectively), overall incidence of blepharoptosis was
• Randomized adult patients with moderate-to- 2.2% in cycle 1 and 0.8% in cycle 2.
severe glabellar lines at maximum frown

44
BoNT/A: Key Efficacy Studies for Periorbital
Wrinkles
► Periorbital wrinkles are the most recent aesthetic indication for
incobotulinumtoxinA and onabotulinumtoxinA.
► The key publications supporting the efficacy of these products in
treating periorbital wrinkles for each BoNT/A agent follow.

45
Key Efficacy Studies for Periorbital Wrinkles:
IncobotulinumtoxinA
The key studies for incobotulinumtoxinA are shown below.
Study Design Conclusions
Prager W et al. • Double-blind, randomized, proof-of-concept • Both botulinum toxin A products displayed high efficacy
Dermatol Surg. study and good tolerability at a dose ratio of 1:1, with no
2010;36 Suppl 4: • Adults with moderate-to-severe bilateral statistically significant differences between them.
2155-2160. symmetrical crow’s feet lines at maximum smile • The high response rates observed after 4 months
suggest good effectiveness beyond this observation
period.
Muti G, • Single-center, randomized, prospective, split- • IncobotulinumtoxinA and onabotulinumtoxinA
Harrington L. face, subject- and rater-blinded study with a (1:1 dose conversion ratio) were well tolerated, showing
Dermatol Surg. clinical crossover evaluation to assess comparable efficacy and duration of treatment effect
2015;41 Suppl 1: incobotulinumtoxinA and onabotulinumtoxinA for crow's feet.
S39-S46. • Adults with symmetrical periorbital wrinkles

46
Key Efficacy Studies for Periorbital Wrinkles:
OnabotulinumtoxinA
The key studies for onabotulinumtoxinA are shown below.
Study Design Conclusions
Carruthers A et al. • Phase 3, multicenter, double-blind, • All primary and secondary end points were achieved;
Dermatol Surg. randomized, placebo-controlled, statistically significant differences favored onabotulinumtoxinA
2014;40(11): parallel-group study (all comparisons vs placebo).
1181-1190. • Adults with moderate-to-severe bilateral • Treatment of moderate-to-severe crow’s feet lines with
symmetrical crow’s feet lines at onabotulinumtoxinA was effective and well tolerated.
maximum smile
Lowe N et al. J Am • Single-center, prospective, double-blind, • All doses of onabotulinumtoxinA were significantly superior to
Acad Dermatol. randomized, split-face study placebo with no clear dose-response relationship. Benefits of
2002;47(6):834- • Adults with moderate-to-severe bilateral the second injection lasted longer than the first.
840. symmetrical crow’s feet lines during • OnabotulinumtoxinA is safe and effective for treatment of
maximum attempted muscle contraction crow's feet. Benefits are more sustained with repeat treatment.
Lowe N et al. • Multicenter, double-blind, randomized, • All doses of onabotulinumtoxinA resulted in improvements in
Dermatol Surg. placebo-controlled, parallel-group, dose- crow's feet severity compared with placebo.
2005;31:257-262. response study • Dose-dependent treatment effect for efficacy observed; higher
• Adults with moderate-to-severe bilateral doses had an increased magnitude and duration of effect.
symmetrical crow’s feet lines at • OnabotulinumtoxinA is safe and effective in decreasing severity
maximum smile of crow's feet.
47
BoNT/A: Key Safety Data for Glabellar Frown
Lines
► As shown by the key studies, the safety and tolerability of BoNT/A products in
moderate-to-severe glabellar frown lines is well established.
► The table below summarizes the AEs commonly reported with each BoNT/A product.
AbobotulinumtoxinA1 IncobotulinumtoxinA2 OnabotulinumtoxinA3
Common (≥1/100 to <1/10 subjects)
Facial paresis (predominantly Paresthesia
Headache
brow paresis Headache
Eye disorders (asthenopia, eyelid Injection site edema, pain, irritation,
Muscle disorders (elevation of face pain, ecchymosis
edema, lacrimation increase, dry eye,
eyebrow, sensation of heaviness)
muscle twitching) Localized muscle weakness
Very Common (≥1/10 subjects) Eyelid ptosis
Headache Erythema, skin tightness
Injection site reactions Nausea
1. Azzalure. Summary of Product Characteristics. Galderma, 2015. 2. Bocouture. Summary of Product Characteristics. Merz, 2015.
3. Vistabel®. Summary of Product Characteristics. Allergan, 2015. 48
BoNT/A: Key Safety Data for Glabellar Frown
Lines (continued)
► Although we have distinguished between products here, the safety
profile of all 3 BoNT/A products can be regarded as similar.

► Now is a good point to take a break and reflect on what you have
learned so far.
► Have you seen any of these effects in your own practice?
► How do you think they could they be avoided?

49
BoNT/A: Key Safety Data
► All products are contraindicated for use in patients with:1-3
► A known hypersensitivity to BoNT/A or components of the formulations, such as
lactose monohydrate or HSA
► An active infection at the injection site
► Neuromuscular disorders, such as myasthenia gravis

► Use of BoNT/A is also cautioned, or should be avoided, in patients:

► Receiving concomitant treatment with agents that interfere with neuromuscular
transmission
► Who are using concomitant anticholinergic drugs
► Who are known to be pregnant or breastfeeding

► To learn more about avoiding and managing AEs associated with BoNT/A
products, please visit Course C in this module.
1. Bocouture. Summary of Product Characteristics. Merz, 2015. 2. Azzalure. Summary of Product Characteristics. Galderma, 2015.
3. Vistabel. Summary of Product Characteristics. Allergan, 2015. 50
BoNT/A: Key Safety Data for Periorbital
Wrinkles
► The table below summarizes the AEs commonly reported with each BoNT/A product.
Common AEs (≥1/100 to <1/10 subjects)
IncobotulinumtoxinA1 OnabotulinumtoxinA2
Eyelid edema, dry eye Eyelid edema, dry eye
Injection site hematoma Injection site hematoma,a injection site hemorrhagea
In the postmarketing setting, flu-like symptoms and Injection-site hematoma was also a common adverse drug
hypersensitivity reactions, erythema, pruritus, rash (local reaction when glabellar frown lines and periorbital wrinkles
and generalized), and breathlessness have been reported. were treated simultaneously.

► Ways to minimize the risk of complications when injecting BoNT/A products are
covered in Course C: Avoiding and Managing Adverse Events Associated with
Botulinum Toxins.

aProcedure-related AEs
1. Bocouture. Summary of Product Characteristics. Merz, 2015. 2. Vistabel. Summary of Product Characteristics. Allergan, 2015. 51
BoNT/A: Interaction with Facial Muscles and
Nerves
► A detailed understanding of facial anatomy is essential to achieving optimal aesthetic
outcomes.
► The close proximity of facial muscles can make it easy to misplace injections,
especially in the lower face, and unexpected diffusion can also lead to poor aesthetic
outcomes.1
► It is important to appreciate that, just as the effects of botulinum toxin are not
permanent in nature, they are also not permanent in aesthetic medicine.
► The effects of BoNT/A are temporary as, while acetylcholine release is being blocked
by BoNT/A, the neuron is forming new synapses to replace original synapses.
► This is known as sprouting, and is only a temporary recovery process, as the original synapses
eventually regenerate while the sprouts are removed.2

1. Inglefield C et al. Expert Consensus on Complications of Botulinum Toxin and Dermal Filler Treatment. London, UK; Aesthetic Medicine Expert Group, 2014.
2. de Paiva A et al. Proc Natl Acad Sci. 1999;96:3200-3205. 52
BoNT/A: Preterminal Sprouting
► The preterminal motor neurons are
myelinated as shown by the large
yellow arrows.
► However, numerous unmyelinated
sprouts arise in the interstitium
between the muscle fibers, as
shown by the small red arrows.

Holds J et al. Invest Ophthamal Visual Sci. 1990;31:964-967. 53

BoNT/A: Preterminal Sprouting (continued)
► Itis thought that the neural cell adhesion molecule (NCAM), which is
upregulated in paralysed muscles, plays a major role in the growth of
paralysis-induced axonal sprouts.1
► NCAM can be used as a marker for the diffusion of BoNT/A once
injected.
► Experiments using NCAM have shown that there is no significant difference in
the diffusion of toxin to adjacent muscles between abobotulinumtoxinA,
incobotulinumtoxinA, and onabotulinumtoxinA.2

1. Schäfer R1, Wernig A. J Neurocytol. 1998;27:615-624. 2. Carli L et al. Muscle Nerve. 2009;40:374-380. 54
BoNT/A: Repeat Dosing

► Given the duration of effect of BoNT/A injections, many patients will return for repeat
injections to ensure the signs of aging do not reappear.
► Therefore, it is important to understand the possible effects of repeat dosing of BoNT/A to an area that
was treated in the past.
► As the duration of action is generally 3 to 5 months, the period between treatment
sessions should be ≥3 months.1–3
► In a recent retrospective analysis of the use of incobotulinumtoxinA in Europe, the majority of treatment
intervals were ≥5 months.4
► The effects of repeated abobotulinumtoxinA treatment in aesthetic medicine have been
examined retrospectively in a large patient cohort.5
► Results showed no evidence of loss of effectiveness or cumulative AEs with repeated treatments.5
► In addition, the doses applied and the satisfaction rates remained stable with 3 to 5 treatments, and the
rate of AEs decreased after the first treatment.5

1. Bocouture. Summary of Product Characteristics. Merz, 2015. 2. Azzalure. Summary of Product Characteristics. Galderma, 2015. 3. Vistabel. Summary of Product
Characteristics. Allergan, 2015. 4. Pavicic T et al. Clin Cosmet Investig Dermatol. 2015:8;135-142. 5. Rzany B et al. Dematol Surg. 2007;33:S18-S125. 55
BoNT/A: Repeat Dosing (continued)

► An open-label study of incobotulinumtoxinA, where patients could receive 2

to 8 repeated cycles of treatment, showed that repeat dosing was effective.1
► General trend towards higher response rates for up to 8 regular cycles of treatment.
► Rapid onset of treatment effect seen in all cycles, and an apparent further increase in
responder rates was observed in Cycle 8 over previous treatment cycles.
► After completing the 4-month phase 3 randomized trials of
onabotulinumtoxinA, subjects were offered participation in an 8-month
open-label, repeat treatment study to assess the safety of repeated
treatment sessions.2
► Progressive improvement with repeated treatment cycles was noted in all efficacy
measures during the extension period.
► The incidence of blepharoptosis decreased with successive treatments, and
injections did not induce neutralizing antibodies.
1. Rzany B et al. Dermatol Surg. 2013;39:95-103. 2. Carruthers A et al. J Clin Res. 2004;7:1-20. 56
The Science Behind BoNT/A: Summary
You should now have an understanding of the following:
► The historical milestones that occurred in the development of BoNT/A for aesthetic use
► The 3 most widely available BoNT/A products and their aesthetic indications
► The molecular structure of BoNT/A
► The MOA of BoNT/A and how it interacts with the muscles and nerves of the face
► The similarities and differences between the 3 most widely available BoNT/A products
► The key papers reporting BoNT/A efficacy and tolerability when treating glabellar frown lines
and periorbital wrinkles

57