Ứng Dụng Thẩm Mỹ Và Lâm Sàng (Cosmetic and Clinical Applications)


Botulinum Toxins
3. Acidification
1. Binding 2. Endocytosis
4. Translocation of
Light Chain
pH: 5
pH: 5
Figure .Multiple steps in the mechanism of action of botulinum toxin type A. This figure illustrates the steps
in the mechanism of action of type A botulinum toxin. (1) Following binding to SV2 (pink), the 150 kDa core
neurotoxin is internalized into the neuron via a process of endocytosis. (2) The endosome is acidified (3), which
facilitates the translocation of the light chain into the neuronal cytosol (4). Once in the neuronal cytosol the
light chain of type A botulinum toxin (yellow) cleaves the membrane-associated SNAP-25, 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).
wall. The interior of the endosome is acidi-
fied, causing a conformational change in the
neurotoxin protein molecule – which then
allows the light chain to traverse the endoso-
mal wall and enter the neuronal cytosol [32].
Once in the cytosol, the disulphide bond teth-
ering the light chain and heavy chain together
is reduced releasing the light chain [2]
(Figure 3.3).
In the neuronal cytosol, the light chain,
a zinc dependent endoprotease, is free to
interact with its specific substrate [33]. Each
serotype of botulinum toxin has a defined
target protein and a highly specific molec-
ular location for proteolytic cleavage. Each
molecular target is a component of the cel-
lular machinery responsible for the dock-
ing of synaptic vesicles and the calcium-
dependent release of neurotransmitter. These
exocytotic proteins are collectively referred
to as the synaptic NSF attachment recep-
tor (SNARE) complex [108]. The SNARE
complex consists of two discrete compo-
nents: those associated with the vesicle and
those associated with the cell membrane.
Interactions between the vesicle-bound and
membrane-associated SNARE proteins hold
the two in apposition. Upon neuronal depo-
larization, the SNARE proteins sense the
increase in intracellular calcium and respond
by fusing the neurotransmitter vesicle with
the membrane [34]. The membrane-bound
SNARE protein SNAP-25 is the target for
type A neurotoxin, cleaving it near its C-
terminus [35]. SNAP-25 is also the target
for types C and E, with type C toxin addi-
tionally cleaving Syntaxin. Types B, D and
F neurotoxins target the vesicle associated
protein Synaptobrevin [36]. The disruption
of the specific target protein and subse-
quent effect on the SNARE complex pre-
vents the fusion of synaptic vesicles with
the neuronal membrane, significantly reduc-
ing the capability to release neurotransmitter
(Figure 3.4).
Although there is general structural simi-
larity between the core neurotoxin in differ-
ent serotypes of botulinum toxins, significant
differences between them occur primarily in
the heavy chain C-terminal region, account-
ing for the differences in receptors for cellular
recognition and binding [21,22].

3 Botulinum Toxin: From Molecule to Medicine

Syntaxin
Synaptobrevin
SNAP-25
Figure .SNARE Complex. Crystal
structure of a core synaptic fusion
complex at 2.4 ˚A resolution. Source:
Sutton 1998 [109]. Reproduced with
permission of Nature Publishing Group.
The duration of botulinum neurotoxin
effect
is
influenced
by
several
factors,
including serotype [37, 38] and somatic vs.
autonomic site of action [3, 5, 39]. The long
duration of effect following a single focal
application of type A botulinum toxin is likely
related to two factors. Firstly, the cleaved
SNAP-25 likely escapes the normal degra-
dation pathway for damaged proteins. The
portion of the SNAP-25 molecule cleaved
by the type A light chain is large enough to
disrupt normal exocytosis, but small enough
that the SNARE complex is preserved and the
cell likely does not recognize that the protein
has been damaged [40]. Secondly, the light
chain of type A toxin appears to evade detec-
tion by cellular sentinels by ‘hiding’ close to
the cell membrane [41]. These properties are
in sharp contrast to the short-acting type
E botulinum toxin. The type E light chain
displays a lack of discrete localization in cells
and distributed throughout the cytoplasm,
making it more vulnerable to degradation
[41] Furthermore, type E cleaves SNAP-25
at a more upstream (proximal) location
than type A, taking a bigger ‘bite’ out of the
molecule, destabilizing the SNARE complex
[2] and allowing the E-cleaved SNAP-25 to
be more readily targeted for degradation
[38,41–43].
The
effects
of
botulinum
toxin
are
reversible, and recovery from denervation
is gradual. It has been demonstrated, in
animal models, that denervation stimulates
the muscle to produce growth factors, such
as insulin-like growth factor-1 (IGF-1) [44],
that trigger the sprouting of new branches
from the silent motor neuron. These new
branches make contact with the dener-
vated muscle, establish new neuromuscular
junctions and become active, contributing
to the restoration of muscle function [45]
while the originally affected neuron recovers
[46]. Full recovery of neuromuscular trans-
mission is not regained until the affected
nerve terminals regain their activity. The
actual persistence time of catalytically active
light chain in motor neurons, in vivo, is
currently unknown, although, in cell culture,
cleaved SNAP-25 has been found 80 days
postexposure to type A botulinum toxin [47].
Following
clinical
administration
of
botulinum toxin type A into striated muscle,
the duration of focal muscle weakening
is typically on the order of 12–16 weeks
[48, 49], while muscle and glands under
autonomic control generally remain inhib-
ited for 26 weeks or longer [3, 5]. The
clinical
effectiveness
of
one
botulinum
toxin product (onabotulinumtoxinA) has


Botulinum Toxins
been demonstrated to persist for at least 6
months following intra-detrusor application
in patients with neurogenic detrusor over-
activity [50] or when injected intradermally
in individuals with severe primary axillary
hyperhidrosis [5]. This may reflect different
metabolic
characteristics
of
autonomic
cholinergic neurons allowing the active light
chain to persist longer than in somatic motor
neurons. Alternatively, it may reflect a lack of
regenerative nerve sprouting in these tissues.
The latter hypothesis has some support from
studies of human bladder in which axonal
sprouting was only rarely observed following
botulinum toxin treatment [51].
Botulinum neurotoxins act to reduce exo-
cytosis from somatic nerves at both of the
locations where they innervate striated mus-
cle. Alpha motor neurons directly contact
the muscle fibers, triggering muscle con-
traction when activated, while A-gamma
motor neurons innervate the muscle spin-
dles. Muscle spindles are intrafusal muscle
fibers responsible for setting the degree of
tone and stretch sensitivity of skeletal mus-
cle [52]. As A-gamma neurons also release
acetylcholine at their peripheral terminals,
botulinum toxin will reduce neurotransmis-
sion at this synapse, modulating the output
of the spindle afferents and thus the level
of muscle tone [53–55], perhaps augmenting
the induced relaxation of the injected muscle
[56]. At least some muscles of facial expres-
sion are reported to lack muscle spindles [57];
therefore, the contribution of this effect in the
aesthetic applications of botulinum toxin in
the face is unclear.
Beyond
the
neuromuscular
junction,
botulinum toxins are effective in treating
neuroglandular overactivity. Several condi-
tions such as hyperhidrosis of the axillae [5]
and palms [59] and sialorrhea [59] have been
demonstrated to be responsive to botulinum
toxin following intradermal injection in the
case of hyperhidrosis or directly into the
salivary glands in the case of sialorrhea. This
suggests that the exocytosis of acetylcholine
at these post-ganglionic autonomic neurons
can also be inhibited by botulinum toxin
treatment.
Sensory Mechanism of Action
A role for botulinum toxin in pain reduc-
tion was proposed following early trials in
cervical dystonia [60–63], and efficacy has
subsequently been investigated in a number
of other painful conditions such as chronic
migraine [64], neuropathic pain [65], myofas-
cial pain [66] and lateral epicondylitis [67].
Although several botulinum toxin products
have been reported to reduce the pain asso-
ciated with cervical dystonia, only onabo-
tulinumtoxinA is approved for the treatment
of pain associated with chronic migraine
[66].
Recent years have brought a significant
increase in the understanding of the mech-
anisms by which botulinum toxin reduces
the perception of pain. Results of a num-
ber of clinical and preclinical studies have
demonstrated that the highly selective dis-
ruption of exocytosis by botulinum toxin
also occurs in the sensory system [69–74].
Botulinum toxin type A was shown to block
the stimulated release of neuropeptide calci-
tonin gene-related peptide (CGRP) from cul-
tured trigeminal ganglion neurons (TGNs)
and substance P from both rat dorsal root
ganglion neurons and TGNs [69, 71]. These
proinflammatory neuropeptides are released
from the peripheral terminals of nocicep-
tive neurons using the SNARE proteins in
response to stimulation, and are known to
also trigger the release of other inflamma-
tory mediators such as bradykinin, glutamate,
prostaglandins and histamine [75]. Periph-
erally administered botulinum toxin type A
significantly attenuated the release of gluta-
mate in rat hindpaw in a dose-dependent
manner following formalin administration
[70,76,77].
In addition, several human models have
demonstrated
that
pretreatment
with
botulinum toxin type A can reduce the flare,
wheal, and cutaneous allodynia associated
with the topical application of capsaicin or
ultraviolet light [78].
More recently, it has been shown that
transport of the transient receptor poten-
tial vanilloid type 1 (TRPV1) channel, to the

3 Botulinum Toxin: From Molecule to Medicine

cell membrane is significantly reduced by
botulinum toxin type A [79,80].
TRPV1 is an ion channel activated by
several classes of painful stimuli and its
expression is known to be upregulated in
chronic pain and inflammation. Thus, the
local effects of botulinum toxin on the
peripheral components of the nociceptive
system may reduce peripheral sensitization
and consequently reduce central sensitiza-
tion through the reduction in the release of
algogenic chemical mediators or by altering
the sensitivity of nociceptive neurons [81]. It
is important to note that neither the large,
myelinated (A-beta) nerve fibers that medi-
ate light touch, vibration and proprioceptive
sensation, nor the lightly myelinated noci-
ceptive fibers (A-delta) that mediate acute
pain appear to be affected by botulinum toxin
type A and therefore botulinum toxin does
not alter proprioception or induce cutaneous
anesthesia or interfere with the normal per-
ception of acute pain [82].
Retrograde Axonal Transport
The profound and long lasting effects of
botulinum toxin on muscle tone in dysto-
nia and spasticity, and the demonstration that
treating cervical dystonia with botulinum
toxin can result in a reorganization of cor-
tical sensory-motor integration [83, 84], has
led some to postulate that there may be a
direct central effect of botulinum toxin within
the central nervous system (CNS) [85]. Prece-
dent for this phenomenon exists by analogy,
as tetanus neurotoxin (also a clostridial neu-
rotoxin that targets SNARE proteins) is ret-
rogradely transported in motor neurons from
the periphery to the spinal cord where it tran-
scytoses to central interneurons, blocking
the release of the central neurotransmitters
GABA and glycine [86]. A single experiment
using an extremely high dose of botulinum
toxin type A has purported to show a sim-
ilar phenomenon in a rat whisker model
[85]. It is possible that, at non-clinical doses
in animal model experiments, botulinum
toxin may overwhelm the normal handling
mechanism and be non-specifically trafficked
to the neuronal cell body, likely for lysosomal
degradation. However, there remains yet no
clear evidence for the transfer of the neuro-
toxin protein from a primary motor neuron
to a central neuron.
There is general consensus that at doses
used in therapeutic or aesthetic applications
of botulinum toxin type A, there is unlikely
to be any direct central effect of the neuro-
toxin [87–89]. Supporting this consensus are
the observations that clinical botulism, char-
acterized by exposure to massive amounts of
botulinum toxin, is vastly different in pre-
sentation than clinical tetanus, with botulism
producing a flaccid paralysis characteris-
tic of a lower motor neuron effect, while
tetanus presents as a spastic paralysis char-
acteristic of an upper motor neuron effect
[90], suggesting that botulinum toxin does
not gain access to central neurons through
transcytocis.
Immunogenicity
As with all protein biologics, the injection of
botulinum toxin may result in activation of
the immune system and the formation of anti-
bodies [91, 92]. While any component of the
botulinum toxin molecule may elicit antibody
formation, only those antibodies that block
the biological activity of the neurotoxin (neu-
tralizing antibodies) will lead to treatment
failure if present in sufficient amounts [93].
A meta-analysis reported that the incidence
of neutralizing antibody formation following
treatment with onabotulinumtoxinA in clini-
cal trials was less than 0.3% [94].
The classification of botulinum toxins by
serotype is an immunologic categorization,
and therefore it stands to reason that neutral-
izing antibodies produced against a product
of one serotype will block responses to other
products of the same serotype. Conversely,
treatment with another serotype may be
employed in cases of true antibody-mediated
non-response [95].
As compared to most protein biologics, the
amount of protein injected during treatment


Botulinum Toxins
with any of the currently approved type A
botulinum toxins is extremely low. For exam-
ple, a 20 U dose of onabotulinumtoxinA con-
tains 1 ng of neurotoxin complex and is usu-
ally administered every 4 months, whereas
the monoclonal antibody adalimumab, used
to treat chronic plaque psoriasis, is typi-
cally administered at a dose of 40 mg twice a
month [96]. On a yearly basis this is 3 ng ver-
sus 960 mg load or a difference of 3,200,000-
fold in dose by weight. The extremely small
amounts of botulinum neurotoxin protein
administered may account for the relatively
low rate of antibody-induced treatment fail-
ure observed with botulinum toxin type A
products. It is also important to note that
some botulinum toxin products may con-
tain residual bacterial contaminants [97] that
might increase the likelihood of developing
an immune reaction.
Products and Pharmacology
Following the first approval of onabotulinum-
toxinA for clinical use by the US FDA in 1989,
several other botulinum toxin type A prod-
ucts have become available. The most widely
known other products are abobotulinumtox-
inA (Ipsen, Ltd, first approved in the UK
in 1990) and incobotulinumtoxinA (Merz,
GmbH, first approved in Germany in 2005).
Although the biological activity of all of these
products resides in a common 150 kD core
neurotoxin, they have differing pharmaco-
logic profiles [98]. Each of these products has
a unique molecular structure, a different mix
of excipients and different definition of a unit,
based on proprietary methodologies for con-
ducting the LD50 assay used to define a unit
of biological activity. These differences are
outlined in the Table 3.1.
Table .Summary of differences between botulinum neurotoxin type A products.
Non-proprietary
name
onabotulinumtoxinA
abobotulinumtoxinA
incobotulinumtoxinA
Manufacturer
Allergan Inc
Ipsen Ltd
Merz GmbH
Molecular weight
(kDa)
∼900 [68]
<500 [99]
150 [100]
Excipients
HSAa 500 μg (100 U vial)
NaClb 0.9 mg
HSA 125 μg (500 U vial)
Lactose 2.5 mg
HSA 1000 μg (100 U vial)
Sucrose 5 mg
Potency assay
Diluent
Cell-based potency assay
Saline [101]
Mouse LD50
Gelatin phosphate [102]
Mouse LD50
Non-saline stabilizer
[103]
150 kDa neurotoxin
(ng)
0.73 (100 U vial) [108]
3.24 (500 U vial) [108]
0.44 (100 U vial) [108]
FDA approved
clinical indications
Cervical dystonia
Blepharospasm
Axillary hyperhidrosis
Upper limb spasticity
Lower limb spasticity
Chronic migraine
Neurogenic detrusor
Overactivity
Idiopathic overactive
bladder
Strabismus
Glabellar Lines
Crow’s feet lines
Cervical dystonia
Upper limb spasticity
Glabellar lines
Cervical dystonia
Blepharospasm in
patients previously
treated with
onabotulinumtoxinA
Upper limb spasticity
Glabellar lines
a HSA, human serum albumin.
b NaCl, sodium chloride.

3 Botulinum Toxin: From Molecule to Medicine

It is important to note that, unlike some
other biologics such as insulin, erythropoi-
etin, vaccines, and many other agents, there
is no international reference preparation for
botulinum toxin products [104] and there-
fore no “International unit” (i.u.). Therefore,
the definition of a unit is not a standard-
ized measure and the units and potency of
each product is independent of all others.
Potency is determined in relation to an in-
house reference standard held by each man-
ufacturer and using their unique and pro-
prietary LD50 assay. Significant differences
are reported to exist in, for example, dilu-
ents used to perform the potency assay: Aller-
gan uses a saline diluent [101], Ipsen use a
gelatin phosphate diluent [102], while Merz
utilizes undefined stabilizers [103] in the dilu-
ent used to define the potency of incobo-
tulinumtoxinA. Testing of onabotulinumtox-
inA and incobotulinumtoxinA using different
assay methodologies has demonstrated dif-
ferences in how each manufacturer defines a
unit, as would be expected given the differ-
ences reported in assays and reference stan-
dards and the differing amounts of core neu-
rotoxin protein in each product. Recognition
of these differences is reflected in each prod-
uct label in the US [68, 100, 105, 106] and in
other countries, and forms the basis for the
unique non-proprietary names for each FDA
approved botulinum toxin product [101].
Allergan has developed a novel cell-based
potency assay as an alternative methodol-
ogy for release testing of onabotulinum-
toxinA. This methodology is specific to
onabotulinumtoxinA, highly sensitive, and
extensively cross-validated against the LD50
assay and was approved by the US FDA
in 2011 and subsequently in most markets
globally.
Due to the complex interplay of manufac-
turing, formulation and potency definition,
there are no generics or biosimilars in this
class of drug. All botulinum toxin products
should be considered unique and not inter-
changeable, and should be dosed and injected
according to the manufacturers recommen-
dations.
Summary
In this chapter we provide a brief overview
of the known mechanisms of action of
botulinum toxin. Botulinum toxin has been
shown, in clinical and preclinical studies, to
modulate vesicular release of neurotransmit-
ters at both alpha and gamma neurons within
the somatic motor system, producing consis-
tent, repeatable benefits in conditions of mus-
cular overactivity. Additionally, effects and
clinical utility for botulinum toxins have been
demonstrated in the autonomic motor sys-
tem and the peripheral nociceptive system.
The specificity of the effect of botulinum
toxins on vesicular neurotransmitter release
forms the basis for the wide variety of doc-
umented clinical applications of this agent.
With a greater depth of understanding of
the structure and pharmacology of botulinum
toxins combined with increased insight into
the workings of the nervous system, it is
likely that the range of clinical indications for
botulinum toxin will continue to grow in the
future.
References
Sudhof TC. The synaptic vesicle cycle.
Annu Rev Neurosci 2004;27:509–547.
Montal M. Botulinum neurotoxin: a
marvel of protein design. Annu Rev
Biochem 2010;79:591–617.
Dmochowski R, Chapple C, Nitti VW,
et al. Efficacy and safety of
onabotulinumtoxinA for idiopathic
overactive bladder: a double-blind,
placebo controlled, randomized, dose
ranging trial. J Urol 2010;184:2416–2422.
Aurora SK, Dodick DW, Turkel CC, et al.
OnabotulinumtoxinA for treatment of
chronic migraine: results from the


Botulinum Toxins
double-blind, randomized,
placebo-controlled phase of the
PREEMPT 1 trial. Cephalalgia
2010;30:793–803.
Lowe NJ, Glaser DA, Eadie N, et al.
Botulinum toxin type A in the treatment
of primary axillary hyperhidrosis: a
52-week multicenter double-blind,
randomized, placebo-controlled study of
efficacy and safety. J Am Acad Dermatol
2007;56:604–611.
Sobel J, Tucker N, Sulka A, McLaughlin J,
Maslanka S. Foodborne botulism in the
United States, 1990–2000. Emerg Infect
Dis 2004;10:1606–1611.
Hatheway CL. Clostridium-botulinum
and other clostridia that produce
botulinum neurotoxin. In: A. H. W.
Hauschild and K. L. Dodds (eds).
Clostridium Botulinum: Ecology and
Control in Foods. New York: Marcel
Dekker, Inc. 1993.
Comella CL, Jankovic J, Shannon KM,
et al. Comparison of botulinum toxin
serotypes A and B for the treatment of
cervical dystonia. Neurology 2005;65:
1423–1429.
Aoki KR. A comparison of the safety
margins of botulinum neurotoxin
serotypes A, B, and F in mice. Toxicon
2001;39:1815–1820.
Johnson EA, Bradshaw M. Clostridium
botulinum and its neurotoxins: a
metabolic and cellular perspective.
Toxicon 2001;39:1703–1722.
Popoff MR, Marvaud JC. Structural and
genomic features of clostridial
neurotoxins. In: J. E. Alouf and J. H. Freer
(eds). The Comprehensive Source Book of
Bacterial Protein Toxins. London:
Academic Press 1999.
Smith TJ, Lou J, Geren IN, et al. Sequence
variation within botulinum neurotoxin
serotypes impacts antibody binding and
neutralization. Infect Immun 2005;73:
5450–5457.
Swaminathan S. Molecular structures and
functional relationships of botulinum
neurotoxins. In: H. Jankovic, A. Albanese,
M. Z. Atassi, et al. (eds). Botulinum Toxin:
Therapeutic Clinical Practise and Science.
Philadelphia: Saunders Elsevier 2009.
Oguma K, Fujinaga Y, Inoue K. Structure
and function of Clostridium botulinum
toxins. Microbiol Immunol 1995;39:
161–168.
Chen F, Kuziemko GM, Stevens RC.
Biophysical characterization of the
stability of the 150-kilodalton botulinum
toxin, the nontoxic component, and the
900-kilodalton botulinum toxin complex
species. Infect Immun 1998;66:
2420–2425.
Hanson MA, Stevens RC. Structural view
of botulinum neurotoxin in numerous
functional states. In: M. F. Brin, J. Jankovic
and M. Hallett (eds). Scienfitic and
Therapeutic Aspects of Botulinum Toxin.
Philadelphia: Lippincott Williams &
Wilkins 2002.
Sharma SK, Singh BR. Enhancement of
the endopeptidase activity of purified
botulinum neurotoxins A and E by an
isolated component of the native
neurotoxin associated proteins.
Biochemistry 2004;43:4791–4798.
Ohishi I, Sugii S, Sakaguchi G. Oral
toxicities of Clostridium botulinum toxins
in response to molecular size. Infect
Immun 1977;16:107–109.
Brandau DT, Joshi SB, Smalter AM, et al.
Stability of the Clostridium botulinum
type A neurotoxin complex: an empirical
phase diagram based approach. Mol
Pharm 2007;4:571–582.
Sakaguchi G, Kozaki S, I. O. Structure and
function of botulinum toxins. In: J. E.
AIouf (eds). Bacterial Protein Toxins.
London: Academic Press. 1984.
Lacy DB, Stevens RC. Sequence homology
and structural analysis of the clostridial
neurotoxins. J Mol Biol 1999;291:
1091–1104.
Rummel A, Hafner K, Mahrhold S, et al.
Botulinum neurotoxins C, E and F bind
gangliosides via a conserved binding site
prior to stimulation-dependent uptake
with botulinum neurotoxin F utilising the

3 Botulinum Toxin: From Molecule to Medicine

three isoforms of SV2 as second receptor.
J Neurochem 2009;110:1942–1954.
Brunger AT, Rummel A. Receptor and
substrate interactions of clostridial
neurotoxins. Toxicon 2009;54:550–560.
Rossetto O, Montecucco C. Peculiar
binding of botulinum neurotoxins. ACS
Chem Biol 2007;2:96–98.
Dong M, Yeh F, Tepp WH, et al. SV2 is the
protein receptor for botulinum
neurotoxin A. Science 2006;312:592–596.
Hughes R, Whaler BC. Influence of
nerve-ending activity and of drugs on the
rate of paralysis of rat diaphragm
preparations by Cl. botulinum type A
toxin. J Physiol 1962;160:221–233.
Wan QF, Zhou ZY, Thakur P, et al. SV2
acts via presynaptic calcium to regulate
neurotransmitter release. Neuron
2010;66:884–895.
Janz R, Goda Y, Geppert M, Missler M,
Sudhof TC. SV2A and SV2B function as
redundant Ca2+ regulators in
neurotransmitter release. Neuron
1999;24:1003–1016.
Jahn R. Neuroscience. A neuronal
receptor for botulinum toxin. Science
2006;312:540–541.
Brunger AT, Jin R, Breidenbach MA.
Highly specific interactions between
botulinum neurotoxins and synaptic
vesicle proteins. Cell Mol Life Sci
2008;65:2296–2306.
Jacky BP, Garay PE, Dupuy J, et al.
Identification of fibroblast growth factor
receptor 3 (FGFR3) as a protein receptor
for botulinum neurotoxin serotype A
(BoNT/A). PLoS pathogens 2013;9(5):
e1003369.
Lai B, Agarwal R, Nelson LD,
Swaminathan S, London E. Low
pH-induced pore formation by the T
domain of botulinum toxin type A is
dependent upon NaCl concentration.
J Membr Biol 2010;236:191–201.
Montal M. Translocation of botulinum
neurotoxin light chain protease by the
heavy chain protein-conducting channel.
Toxicon 2009;54:565–569.
Jahn R, Sudhof TC. Membrane fusion and
exocytosis. Annu Rev Biochem 1999;68:
863–911.
Blasi J, Chapman ER, Link E, et al.
Botulinum neurotoxin A selectively
cleaves the synaptic protein SNAP-25.
Nature 1993;365:160–163.
Simpson LL. Identification of the major
steps in botulinum toxin action. Annu Rev
Pharmacol Toxicol 2004;44:167–193.
Eleopra R, Tugnoli V, Rossetto O, De
Grandis D, Montecucco C. Different time
courses of recovery after poisoning with
botulinum neurotoxin serotypes A and E
in humans. Neurosci Lett 1998;256:
135–138.
Foran PG, Mohammed N, Lisk GO, et al.
Evaluation of the therapeutic usefulness of
botulinum neurotoxin B, C1, E, and F
compared with the long lasting type A.
Basis for distinct durations of inhibition of
exocytosis in central neurons. J Biol Chem
2003;278:1363–1371.
Maia FM, Kanashiro AK, Chien HF,
Goncalves LR, Barbosa ER. Clinical
changes of cervical dystonia pattern in
long-term botulinum toxin treated
patients. Parkinsonism Relat Disord
2010;16:8–11.
Bajohrs M, Rickman C, Binz T, Davletov
B. A molecular basis underlying
differences in the toxicity of botulinum
serotypes A and E. EMBO Rep 2004;5:
1090–1095.
Fernandez-Salas E, Steward LE, Ho H,
et al. Plasma membrane localization
signals in the light chain of botulinum
neurotoxin. Proc Natl Acad Sci U S A
2004;101:3208–3213.
Dolly JO, Aoki KR. The structure and
mode of action of different botulinum
toxins. Eur J Neurol 2006;13 Suppl 4:1–9.
Rickman C, Meunier FA, Binz T, Davletov
B. High affinity interaction of syntaxin
and SNAP-25 on the plasma membrane is
abolished by botulinum toxin E. J Biol
Chem 2004;279:644–651.
Shen J, Ma J, Lee C, et al. How muscles
recover from paresis and atrophy after


Botulinum Toxins
intramuscular injection of botulinum
toxin A: Study in juvenile rats. J Orthop
Res 2006;24:1128–1135.
Rogozhin AA, Pang KK, Bukharaeva E,
Young C, Slater CR. Recovery of mouse
neuromuscular junctions from single and
repeated injections of botulinum
neurotoxin A. J Physiol 2008;586:
3163–3182.
de Paiva A, Meunier FA, Molgo J, Aoki
KR, Dolly JO. Functional repair of motor
endplates after botulinum neurotoxin type
A poisoning: biphasic switch of synaptic
activity between nerve sprouts and their
parent terminals. Proc Natl Acad Sci USA
1999;96:3200–3205.
Keller JE, Neale EA, Oyler G, Adler M.
Persistence of botulinum neurotoxin
action in cultured spinal cord cells. FEBS
Lett 1999;456:137–142.
Brashear A, Watts MW, Marchetti A,
et al. Duration of effect of botulinum
toxin type A in adult patients with cervical
dystonia: a retrospective chart review.
Clin Ther 2000;22:1516–1524.
Naumann M, Yakovleff A, Durif F. A
randomized, double-masked, crossover
comparison of the efficacy and safety of
botulinum toxin type A produced from
the original bulk toxin source and current
bulk toxin source for the treatment of
cervical dystonia. J Neurol 2002;249:
57–63.
Herschorn S, Gajewski J, Ethans K, et al.
Efficacy of botulinum toxin A injection for
neurogenic detrusor overactivity and
urinary incontinence: a randomized,
double-blind trial. J Urol 2011;
Haferkamp A, Schurch B, Reitz A, et al.
Lack of ultrastructural detrusor changes
following endoscopic injection of
botulinum toxin type a in overactive
neurogenic bladder. Eur Urol 2004;46:
784–791.
Hulliger M. The mammalian muscle
spindle and its central control. Rev Physiol
Biochem Pharmacol 1984;101:1–110.
Trompetto C, Curra A, Buccolieri A, et al.
Botulinum toxin changes intrafusal
feedback in dystonia: a study with the
tonic vibration reflex. Mov Disord
2006;21:777–782.
Trompetto C, Bove M, Avanzino L, et al.
Intrafusal effects of botulinum toxin in
post-stroke upper limb spasticity. Eur J
Neurol 2008;15:367–370.
Rosales RL, Arimura K, Takenaga S,
Osame M. Extrafusal and intrafusal
muscle effects in experimental botulinum
toxin-A injection. Muscle Nerve 1996;19:
488–496.
Filippi GM, Errico P, Santarelli R, Bagolini
B, Manni E. Botulinum A toxin effects on
rat jaw muscle spindles. Acta Otolaryngol
1993;113:400–404.
Goodmurphy CW, Ovalle WK.
Morphological study of two human facial
muscles: orbicularis oculi and corrugator
supercilii. Clin Anat 1999;12:1–11.
Saadia D, Voustianiouk A, Wang AK,
Kaufmann H. Botulinum toxin type A in
primary palmar hyperhidrosis:
randomized, single-blind, two-dose study.
Neurology 2001;57:2095–2099.
Lagalla G, Millevolte M, Capecci M,
Provinciali L, Ceravolo MG. Botulinum
toxin type A for drooling in Parkinson’s
disease: a double-blind, randomized,
placebo-controlled study. Mov Disord
2006;21:704–707.
Tsui JK, Fross RD, Calne S, Calne DB.
Local treatment of spasmodic torticollis
with botulinum toxin. Can J Neurol Sci
1987;14:533–535.
Poewe W, Schelosky L, Kleedorfer B, et al.
Treatment of spasmodic torticollis with
local injections of botulinum toxin.
One-year follow-up in 37 patients.
J Neurol 1992;239:21–25.
Odergren T, Tollback A, Borg J. Efficacy of
botulinum toxin for cervical dystonia. A
comparison of methods for evaluation.
Scand J Rehabil Med 1994;26:191–195.
Brin et al. Movement Disorders Vol 2(4)
p237–254.
Dodick DW, Turkel CC, DeGryse RE,
et al. OnabotulinumtoxinA for treatment
of chronic migraine: pooled results from

3 Botulinum Toxin: From Molecule to Medicine

the double-blind, randomized,
placebo-controlled phases of the
PREEMPT clinical program. Headache
2010;50:921–936.
Ranoux D, Attal N, Morain F, Bouhassira
D. Botulinum toxin type A induces direct
analgesic effects in chronic neuropathic
pain. Ann Neurol 2008;64:274–283.
Foster L, Clapp L, Erickson M, Jabbari B.
Botulinum toxin A and chronic low back
pain: a randomized, double-blind study.
Neurology 2001;56:1290–1293.
Placzek R, Drescher W, Deuretzbacher G,
Hempfing A, Meiss AL. Treatment of
chronic radial epicondylitis with
botulinum toxin A. A double-blind,
placebo-controlled, randomized
multicenter study. J Bone Joint Surg Am
2007;89:255–260.
BOTOX® (onabotulinumtoxinA)
Prescribing Information. Allergan, Inc.
Irvine, CA, October, 2010.
Durham PL, Cady R, Cady R. Regulation
of calcitonin gene-related peptide
secretion from trigeminal nerve cells by
botulinum toxin type A: implications for
migraine therapy. Headache
2004;44:35–42;discussion 42–33.
Cui M, Khanijou S, Rubino J, Aoki KR.
Subcutaneous administration of
botulinum toxin A reduces
formalin-induced pain. Pain 2004;107:
125–133.
Purkiss J, Welch M, Doward S, Foster K.
Capsaicin-stimulated release of substance
P from cultured dorsal root ganglion
neurons: involvement of two distinct
mechanisms. Biochem Pharmacol
2000;59:1403–1406.
Gazerani P, Au S, Dong X, et al.
Botulinum neurotoxin type A (BoNTA)
decreases the mechanical sensitivity of
nociceptors and inhibits neurogenic
vasodilation in a craniofacial muscle
targeted for migraine prophylaxis. Pain
2010;151:606–616.
Gazerani P, Staahl C, Drewes AM,
Arendt-Nielsen L. The effects of
botulinum toxin type A on
capsaicin-evoked pain, flare, and
secondary hyperalgesia in an
experimental human model of trigeminal
sensitization. Pain 2006;122:315–325.
Gazerani P, Staahl C, Drewes AM,
Arendt-Nielsen L. Botulinum toxin type A
reduces capsaicin-evoked sensory and
vasomotor responses in human skin.
Neurotox Res 2006;9: ABS-P13.
McMahon SB, Bennett DLH, Bevan
Bennett S. Inflammatory mediators and
modulators of pain. In: S. McMahon and
M. Koltzenburg (eds). Wall and Melzack’s
Textbook of Pain. New York: Churchill
Livingstone 2005.
Gazerani P, Pedersen NS, Staahl C,
Drewes AM, Arendt-Nielsen L. Pain 2009
Jan;141(1–2):60–9.
Tugnoli V, Capone JG, Eleopra R,
Quatrale R, Sensi M, Gastaldo E, Tola MR,
Geppetti P. Pain 2007 Jul;130(1–2):76–83.
The UVB cutaneous inflammatory pain
model: a reproducibility study in healthy
volunteers. Carsten Dahl Mørch, Parisa
Gazerani, Thomas A Nielsen, Lars
Arendt-Nielsen. Int J Physiol Pathophysiol
Pharmacol 2013;5(4):203–215. Published
online 2013 December 15.
TNFα induces co-trafficking of
TRPV1/TRPA1 in VAMP1-containing
vesicles to the plasmalemma via
Munc18–1/syntaxin1/SNAP-25 mediated
fusion. Meng J, Wang J, Steinhoff
M, Dolly JO. Sci Rep 2016 Feb 18;6:21226.
doi: 10.1038/srep21226.
Morenilla-Palao C, Planells-Cases R,
Garcia-Sanz N, Ferrer-Montiel A.
Regulated exocytosis contributes to
protein kinase C potentiation of vanilloid
receptor activity. J Biol Chem 2004;279:
25665–25672.
Aoki KR. Review of a proposed
mechanism for the antinociceptive action
of botulinum toxin type A.
Neurotoxicology 2005;26:785–793.
Gazerani P, Pedersen NS, Staahl C,
Drewes AM, Arendt-Nielsen L.
Subcutaneous Botulinum toxin type A
reduces capsaicin-induced trigeminal pain


Botulinum Toxins
and vasomotor reactions in human skin.
Pain 2009;141:60–69.
Walsh R, Hutchinson M. Molding the
sensory cortex: spatial acuity improves
after botulinum toxin treatment for
cervical dystonia. Mov Disord 2007;22:
2443–2446.
Thickbroom GW, Byrnes ML, Stell R,
Mastaglia FL. Reversible reorganisation of
the motor cortical representation of the
hand in cervical dystonia. Mov Disord
2003;18:395–402.
Antonucci F, Rossi C, Gianfranceschi L,
Rossetto O, Caleo M. Long-distance
retrograde effects of botulinum
neurotoxin A. J Neurosci 2008;28:
3689–3696.
Lalli G, Bohnert S, Deinhardt K,
Verastegui C, Schiavo G. The journey of
tetanus and botulinum neurotoxins in
neurons. Trends Microbiol 2003;11:
431–437.
Curra A, Berardelli A. Do the unintended
actions of botulinum toxin at distant sites
have clinical implications? Neurology
2009;72:1095–1099.
Caleo M, Schiavo G. Central effects of
tetanus and botulinum neurotoxins.
Toxicon 2009;54:593–599.
Caleo M, Antonucci F, Restani L,
Mazzocchio R. A reappraisal of the central
effects of botulinum neurotoxin type A: by
what mechanism? J Neurochem 2009;109:
15–24.
Humeau Y, Doussau F, Grant NJ, Poulain
B. How botulinum and tetanus
neurotoxins block neurotransmitter
release. Biochimie 2000;82:427–446.
Schellekens HH. Immunogenicity of
therapeutic proteins. Clin Ther 2002;24:
1720–1740.
Shankar G, Pendley C, Stein KE. A
risk-based bioanalytical strategy for the
assessment of antibody immune
responses against biological drugs. Nat
Biotechnol 2007;25:555–561.
Goschel H, Wohlfarth K, Frevert J,
Dengler R, Bigalke H. Botulinum A toxin
therapy: neutralizing and nonneutralizing
antibodies – therapeutic consequences.
Exp Neurol 1997;147:96–102.
Naumann M, Carruthers A, Carruthers J,
et al. Meta-analysis of neutralizing
antibody conversion with
onabotulinumtoxinA (BOTOX(R)) across
multiple indications. Mov Disord
2010;25:2211–2218.
Brin MF, Lew MF, Adler CH, et al. Safety
and efficacy of NeuroBloc (botulinum
toxin type B) in type A-resistant cervical
dystonia. Neurology 1999;53:1431–1438.
Humira® (adalimumab) Injection
Prescribing Information. Abbott
Laboratories. North Chicago, IL. March,
2011.
Panjwani N, O’Keeffe R, Pickett A.
Biochemical, functional and potency
characteristics of type A botulinum toxin
in clinical use. Botulinum J 2008;1:
153–166.
Foster KA, Bigalke H, Aoki KR. Botulinum
neurotoxin – from laboratory to bedside.
Neurotox Res 2006;9:133–140.
Hambleton P. Clostridium botulinum
toxins: a general review of involvement in
disease, structure, mode of action and
preparation for clinical use. J Neurol
1992;239:16–20.
Xeomin® (incobotulinumtoxinA)
Prescribing Information. Merz
Pharmaceuticals, LLC. August, 2010.
Schantz EJ, Johnson EA. Properties and
use of botulinum toxin and other
microbial neurotoxins in medicine.
Microbiol Rev 1992;56:80–99.
Hambleton P, Pickett AM. Potency
equivalence of botulinum toxin
preparations. J R Soc Med 1994;87:
719.
Mander G, Fink K, Vey M. Experimental
conditions substantially influence
botulinum toxin potency testing. Clin
Neuropharmacol 2009;32:234;author
reply 235.
Adler S, Bicker G, Bigalke H, et al. The
current scientific and legal status of
alternative methods to the LD50 test for
botulinum neurotoxin potency testing.

3 Botulinum Toxin: From Molecule to Medicine

The report and recommendations of a
ZEBET Expert Meeting. Altern Lab Anim
2010;38:315–330.
Myobloc® (rimabotulinumtoxinB)
Prescribing Information. Solstice
Neurosciences, Inc. South San Francisco,
CA, May, 2010.
Dysport® (abobotulinumtoxinA)
Prescribing Information. Ipsen Biopharm
Ltd., April, 2010.
Lee K, Gu S, Jin L, Le TTN, Cheng LW,
et al. (2013) Structure of a bimodular
botulinum neurotoxin complex provides
insights into its oral toxicity. PLoS Pathog
9(10): e1003690. doi:10.1371/journal
.ppat.1003690
Frevert, J., Content of Botulinum
Neurotoxin in Botox_/Vistabel,
Dysport_/Azzalure and Xeomin_/
Bocouture_ Drugs R D 2010;10 (2):67–73
Sutton, B., Fasshauer, D., Jahn. R.,
Br¨unger. A.T. (1998) Crystal structure of a
core synaptic fusion complex Nature 395,
347–353.



Myobloc
Neil S. Sadick, MD (FACP, FAAD, FAACS, FACPh)1,2 and Suveena Manhas-Bhutani, MD2
1Clinical Professor, Weill Cornell Medical College, Cornell University, New York, USA
2Sadick Dermatology and Research, New York, USA
The use of botulinum toxins for the improve-
ment of fine lines and facial wrinkles has
become increasingly popular over the past
several years. By utilizing toxins to target
hyperfunctional facial rhytids, physicians are
able to successfully treat aging skin, yield-
ing aesthetically younger, smoother looking
skin to patients [1–10]. The occurrence of
glabellar lines is due to the constant pulling
of the skin by the musculature below. Active
muscle movement of the transverse head of
the corrugator supercilii results in the ver-
tical glabellar lines, while the oblique head
of the corrugator supercilii and the depres-
sor supercilii along with the orbicularis oculi
muscles contribute to the formation of the
oblique glabellar line [11]. The approach to
treatment includes minimizing the appear-
ance of the glabellar lines by consider-
ing options of microdermabrasion, chemical
peel, injections of collagen, silicone, autolo-
gous fat or dermal fillers. There are also surgi-
cal venues of endoscopy or limited incision to
modify function of the corrugator supercilii
and procerus muscle and direct incision of
the glabellar line (Figure 4.1 and Figure 4.2).
In 1992, Carruthers and Carruthers eval-
uated the use of Clostridium botulinum
type A purified neurotoxin complex (BTX-
A; Botox®, Allergan Inc., Irvine, CA) to erad-
icate glabellar rhytids by chemically dener-
vating the corrugator supercilii [5]. Findings
included that botulinum toxin blocked the
release of acetylcholine from motor nerve
endings, resulting in a decrease in mus-
cle tone and over-activity that was dose-
dependent. It was also noted that botulinum
toxin provided a safe treatment that targeted
the muscle activity that contributed to the
formation of wrinkle lines. Since then, myriad
of other studies have yielded similar results,
and the BTX-A is currently FDA-cleared for
cosmetic use in moderate to severe glabellar
lines and crow’s feet.
Botulinum toxin type B (BTX-B; Myobloc
in the USA and Neurobloc in Europe; other-
wise identified as Myobloc henceforth, Sol-
stice Neurosciences, South San Francisco,
CA) was FDA-cleared in 2000 for the treat-
ment of abnormal head position and pain
related to cervical dystonia. Currently its clin-
ical utility is being investigated for a variety of
other medical disorders, such as hyperhidro-
sis and siallorhea, as well as in the setting
of cosmetic dermatology. It is readily avail-
able in a liquid formulation, which requires
no preparation or reconstitution unlike BTX-
A [8,12–15].
Pharmacology of Botulinum
Toxin B
The molecular weight of the BTX-A complex
is around 900 kDa whereas that of Myobloc
is
approximately
700 kDa.
Both
these
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:


Botulinum Toxins
SNARE Proteins
Pre-synaptic
nerve
surface
BoNT-
E C A
Acetylcholine
vesicle
BoNT-
D, F
BoNT-C
SYNAPSE
NERVE TERMINAL
SNAP-25
Syntaxin
Synaptobrevin-2 (VAMP)
BoNT-G
BoNT-B
Figure .Pharmacology of botulinum toxins at the neuromuscular junction. SNAP-25 is the target for the type
A toxin (red arrow), synaptobrevin is the target for the type B toxin (black arrow).
complexes are found to be stable in acidic
conditions. The large neurotoxin complex is
composed of proteins such as a hemagglu-
tinin and a non-toxic, non-hemagglutinin
moiety. The active neurotoxin is a 150 Da
dimer consisting of heavy chain (100 kDa)
and a light chain (50 kDa) moiety linked
together by a disulfide bond.
The mechanism of action of botulinum
toxin B on the nervous system is simi-
lar to that of all other botulinum toxins
and involves a series of cellular actions
Figure .Schemata showing the most common sites of botulinum toxin injections in the upper face.
Injections in (a) the glabella; (b) the forehead; (c) the crow’s feet. The author prefers to use the conversion of
150 U of type B toxin for 1 U of type A toxin.

4 Myobloc

including binding, internalization, and inhi-
bition of acetylcholine release (Figure 4.1).
The heavy chain contributes to the irre-
versible binding of the toxin to the serotype-
specific protein within the soluble N-ethyl
maleimide-sensitive factor attachment pro-
tein receptor (SNARE) complex, which is
involved in release of acetylcholine contain-
ing vesicles from the presynaptic neuron.
Flaccid paralysis associated with botulism is
due to the light chain-mediated proteoly-
sis of SNARE proteins and the subsequent
inhibition of synaptic vesicle fusion to the
presynaptic membrane of the human motor
neurons Tis. A specific residue on one of the
N-ethyl maleimide-sensitive factor attach-
ment proteins is cleaved by each serotype.
Myobloc cleaves vesicle associated mem-
brane protein (VAMP), also known as synap-
tobrevin. Serotypes F, G, D also cleave VAMP,
but at various locations. It is found that the
various sites of action of various serotypes
may contribute to the differences in clini-
cal effectiveness. SNARE proteins may be the
only intracellular targets of BTX serotypes.
The remarkable specificity of the BTX light
chain cleavage is due to the light chain rec-
ognizing unusually long SNARE sequences
(∼30–50 residues indicated by the specific
serotype) [16–22] (Table 4.1).
Immunogenicity of Myobloc
The injection of any botulinum toxin may
elicit an immune response since it is con-
sidered a foreign protein injected into the
body [16]. Patients that are treated with
any toxin can develop neutralizing anti-
bodies that blunt clinical effectiveness [23].
When these antibodies are present, the ther-
apeutic effect may be significantly reduced
or completely nullified, based on retrospec-
tive studies by Dresseler and Bigalke [13]
and Dolimbek et al. [6], where 3–5% of
patients were treated with dose a of 100–
1200 U of BTX-A for cervical dystonia. For-
mation of antibodies to other parts of the
complex is not believed to affect clinical
performance of these products. The adverse
Table .Pharmacology of botulinum toxin
serotypes by target/cleavage sizes.
Serotype
Target size
Cleaves at
A
SNAP-25a
Gln197-Arg198
B
VAMPb
Glu76-Phe77
C
Syntaxin
Lys253-Ala254
Lys252-Ala252
SNAP-25a
Arg198-Ala199
D
VAMPb
Ala67-Asp68
Lys59-Leu60
E
SNAP-25a
Arg180-Ile181
F
VAMPb
Gln58-Lys59
G
VAMPb
Ala81-Ala82
aSNAP-25
is
soluble
NSF attachment
protein
of
25,000 kDa.
bVAMP is vesicle-associated membrane protein (synap-
tobrevin).
events are considered significantly lower in
patients treated for aesthetic purposes as
compared to those patients being treated for
cervical dystonia as lower doses are utilized
to treat cosmetic issues [24]. When patients
become resistant to one particular serotype,
another serotype can be used, as botulinum
toxin serotypes are not able to undergo
cross – neutralization. In various studies con-
ducted on patients with cervical dystonia,
those resistant to BTX-A responded to treat-
ment with Myobloc. Furthermore, patients
resistant to Myobloc can achieve positive
results from BTX-A or any other botulinum
toxin serotype. Recent research suggests that
resistance to one serotype may potentiate
resistance to the other following repeat and
multiple injections. Risks associated with
cross-antibody resistance include treatment
with high doses of toxin, increased frequency
of administration, and the high amount of
neurotoxin protein per injection [23–25].
Liquid Formulation of Myobloc
Myobloc is prepared in a ready-to-use for-
mulation, which is stabilized at a pH of
5.6. It may be slightly more painful than


Botulinum Toxins
BTX-A due to its higher level of acidity
[12]. Vials of Myobloc are found in 2,500,
5,000, and 10,000 U, each with concentrations
of 5,000 U/ml or 500 U/0.1 ml [26]. Berman
et al. [24] noted that this preparation can
be diluted up to sixfold without losing its
viability. Although, one unit equates to the
amount of toxin that is detrimental in 50%
of female Swiss–Webster mice after intraperi-
toneal injections (Mouse LD50 bioassay) it
is imperative to understand the sensitivity
of different species to each toxin serotype.
Furthermore, the differences in the potency
assays employed and different toxin prod-
ucts cannot be compared on a unit-by-unit
basis [27]. Specific dose ranging studies in
humans are essential for each toxin serotype
and for each indication, and it is important to
understand that dose conversion should not
be attempted as noted in the package instruc-
tions for BTX -A and Myobloc [24].
Storage and refrigeration of Myobloc is
beneficial at 2–8◦C in a constant environ-
ment for up to 3 years. It is stable for at least
9 months at a room temperature of 25◦C and
remains biologically active when stored in
refrigeration for 21 months and then moved
back to room temperature for 6 months and
then moved to 4◦C. Myobloc diluted up to
sixfold with either non-preserved or pre-
served saline can sustain potency for at least
24 h at room temperature [12,17,26,28,29].
Myobloc in the Aesthetics Practice
Although its primary use for the treatment of
cervical dystonia, increasing demand for cos-
metic improvements such as effective treat-
ment of facial rhytids, have expanded the
off-label use of Botulinum Toxin B to a vari-
ety of other aesthetic indications. Several
peer-reviewed clinical studies and trials have
demonstrated the safety and efficacy of BTX-
B use in aesthetic medicine, mostly focus-
ing on establishing therapeutic protocols and
doses [7].
Alster and Lupton treated 20 female
patients with vertical glabellar rhytids who
had minimal response to BTX-A (less than
50% reduction in contraction of the mus-
cles treated with BTX-A). Subjects were
treated with Myobloc at five standardized
intramuscular sites (procerus, inferomedial
corrugators and superior medial corrugators)
at a total dose of 2,500 U [1]. Improvement
of wrinkles occurred in all individuals with
peak response at 1 month and complete
dissolution of effect at month 4.
Flynn and Clark showed an increased
rate of onset and widespread diffusion of
Myobloc versus BTX-A in a single centre
study of 24 patients with symmetrical, mod-
erate to severe forehead wrinkles [19]. Sub-
jects received type A (BOTOX®) 5 U on one
side and type B (Myobloc®) 500 U on the
other side of the forehead. Measurements of
radius of diffusion and time until full effect
took place were taken. Myobloc was found to
have a slightly faster rate of onset than type
A. A greater radius of diffusion was consis-
tently observed with type B as measured by
the greater area of wrinkle reduction at the
doses used.
Ramirez et al. evaluated Myobloc in 24
subjects with facial wrinkles [31]. Eighty-two
percent had been treated with BTX-A pre-
viously, but not for 6 months prior to the
study. Each subject received 200–400 U of
Myobloc per unilateral injection site (total
dose, 400–800 U). Three sites were injected,
including the frontalis, the corrugators, and
the orbicularis oculi. Improvement in facial
wrinkles was assessed using two scales: the
Wrinkle Improvement Scale (WIS; 0 indi-
cates no improvement and 3 indicates signif-
icant improvement) and the Rated Numeric
Kinetic Line Scale (RNKLS). The latter scale
describes wrinkles both at rest and at max-
imum frown. It ranges from a score of 1,
reflecting no wrinkles at rest that become
fine lines with facial animation, to a score of
4, denoting deep lines at rest that become
deep furrows with facial animation. Subject
response was evaluated before injection and
after injection at visits, that is weeks 1, 2,
4, 8, and 12. Photographs were also docu-
mented. All subjects exhibited rapid onset

4 Myobloc

of target muscle effect within 72 hours and,
in some cases, within 24 hours. Scores on
the WIS and RNKLS were moderately to sig-
nificantly improved by two to three points
after Myobloc treatment; however, the dura-
tion of effect was suboptimal (mean of 8
weeks). No subjects reported side effects of
dysphagia, dyspepsia, or dry mouth, which
are commonly seen in treatment of cervi-
cal dystonia. Eyelid ptosis, brow ptosis, and
dry eye were not measured. This prelimi-
nary study showed that Myobloc is effective
in treating facial lines of the glabella, fore-
head, and crow’s feet areas, but the authors
concluded that doses higher than 400–800 U
would be necessary for a longer duration of
action.
This study also evaluated the subjects for
pain associated with injection, using the
McGill Pain Scale. Pain experienced upon
injection, usually described as a slight sting-
ing sensation, has been reported to occur
with botulinum toxin injections. The McGill
Pain Scale is a validated scale ranging from
0, signifying no pain to 5, signifying excru-
ciating pain. At the time of treatment, sub-
jects were asked to rate the pain of Myobloc
injection and to rate by memory the pain
of BTX-A injection. On average, Myobloc
was found to be slightly more painful than
the memory of BTX-A injection (2.3 vs. 1.6
respectively), although all subjects indicated
that the injection pain would not prevent
them from undergoing a repeat injection with
Myobloc. A recent study by Lowe and Lowe
[32], showed that injection pain of the acidic
solution of BTX-B neurotoxin was reduced
and efficacy not compromised by changing
the pH of BTX-B solution to pH 7.5.
Sadick conducted two open-label studies
using higher doses of BTX-B for treatment of
glabellar wrinkles [10, 30]. Both studies were
similar in design, but the first study evalu-
ated BTX-B 1,800 U (N = 30), and the sec-
ond study evaluated 2,400 U (N = 16) and
3,000 U (N = 18). Doses were divided equally
among six sites; two in the procerus and two
in each corrugator supercilii and orbicularis
oculi muscle bilaterally. Most of the subjects
had not been previously treated with BTX-
A. In the first study, clinical photography and
a clinical scoring system was used by both
subjects and physicians to evaluate efficacy; 0
denoted marked frowning ability, 1 denoted
partial frowning ability, and 2 denoted com-
plete inability to frown because of paralysis.
The second study also used the RNKLS. Sub-
jects returned to the office daily postinjec-
tion until the effects of BTX-B were observed
and weekly thereafter. Both studies found
BTX-B to be effective in treating glabellar
frown lines, based on photography, patient
satisfaction, and improvements in assessment
scores. Overall, BTX-B had a very rapid
onset of action. Comparing the results of
both studies also suggest that the duration of
response is dose related. The mean duration
of effect was 8.0 weeks with 1,800 U, 9.6 weeks
with 2,400 U, and 10.4 weeks with 3,000 U.
Lid ptosis was reported in one patient who
received 2,400 U and in one patient who
received 3,000 U. Headaches and mild pain
upon injection were also reported. Over-
all, BTX-B was very safe, and there was no
increase in adverse effects with the higher
doses.
In a dosing study, Lowe et al. assessed 13
subjects injected with Myobloc in two dif-
ferent dose schedules versus BTX-A in the
corrugator-procerus complex. One subgroup
received a conversion of 50 U of Myobloc
(total 1,000) to 1 U of BTX-A, while oth-
ers received a conversion factor of 100 U of
Myobloc (total 2,000) to 1 U BTX-A. Patients
treated with BTX-A received a total of 20 U.
Both types of botulinum toxins were noted
to be effective in improving glabellar frown
lines. The onset of action was more rapid (2–
3 days) with Myobloc than with BTX-A (3–
7 days). Duration of effect with BTX-A was
at least 16 weeks. While the 1,000 U Myobloc
doses, duration was 6–8 weeks and with the
2,000 U Myobloc doses the duration was 10–
12 weeks [33].
Kim investigated an escalating dosage for-
mula of 400–800 U to 1,600–2,000 U and
2,500–3,000 U per site treating the bilat-
eral frontalis, corrugator or orbicularis oculi


Botulinum Toxins
using a WIS and RNKLS, found Myobloc
to be clinically effective for up to 8 weeks
for treatment of hyperkinetic facial lines
and up to week 12 at the higher dosage
[34]. The patients exhibited excellent skin
smoothing, which was felt to be secondary
to an enhanced diffusion effect of Myobloc.
Reported side effects included headaches
(40%), brow ptosis (7%), dry eyes (5%) and dry
mouth (13%).
It is observed from the various clinical
assessments on Myobloc that the highest
dose to be used for aesthetic means is 3,125 U
for glabellar lines and 3,750 U for the frontalis
region. In an open-label study, 26 subjects
received low (1,875 U), medium (2,500 U), or
high (3,125 U) doses of Myobloc for glabel-
lar lines; 18 subjects received a low (2,250 U),
medium (3,000 U), or high (3,750 U) dose of
Myobloc in the frontalis. Results were sim-
ilar to other studies of Myobloc for wrin-
kles in that it has a very rapid onset of effect
and a dose-related duration of effect. There
were no reports of lid ptosis. Myobloc was
reported to yield a very uniform paralysis and
a smooth aesthetic effect. This effect has also
been observed by the author, as well as by oth-
ers, and may reflect the diffusion characteris-
tics of Myobloc, which may differ from those
of BTX-A. Compared with BTX-A, Myobloc
appears to diffuse more within the injected
muscle [35].
Studies of Myobloc for the treatment of
facial wrinkles demonstrate its effectiveness,
rapid onset of action, and dose-dependent
duration of effect. In clinical studies of cer-
vical dystonia, the calculated duration of the
effect of Myobloc was 12–16 weeks, which is
comparable to that seen with BTX-A. These
trials demonstrated a dose–response rela-
tionship in the duration of effect when treat-
ing glabellar lines. It is anticipated that higher
doses of Myobloc can be safely and effectively
administered to produce an even longer dura-
tion of response in the treatment of facial
wrinkles. Further studies at higher doses are
recommended to determine optimal dosing
of Myobloc, without compromising its safety
profile [36–38].
Treatment Considerations for
Myobloc
Previous studies have been used as a basis
to establish initial approximates for effec-
tive doses of Myobloc in the treatment of
facial rhytids [1, 3–5, 8, 10, 11, 31, 34, 39–52]
(Table 4.2).
The procerus complex and corrugators
supercilii muscles are treated to eradicate
glabellar lines. A dose totaling 20–30 U of
BTX-A is divided and equally injected into
five sites compared to the Myobloc treat-
ment, which requires 2,000–3,000 U divided
among only four injection sites resulting
in comparable results (Figure 4.2). The
injection of the frontalis utilizes 15–30 U of
BTX-A distributed evenly over five to six
injection
sites
and
provides
satisfactory
results while the Myobloc in quantities of
1,000–2,500 U per side yields similar results
as well. To achieve effective results in the
lifting of the brow, injections of 1,500 U are
directed to the corrugator supercilii, the
procerus complex and the medial portion
of the orbicularis oculi muscle. For the
treatment of crow’s feet injecting 10–15 U
of BTX-A divided into two to three sites
per side yields similar results as employ-
ing 1,000–1,500 U per side of Myobloc,
while diffusion characteristics of Myobloc
taken into consideration in this scenario
[4, 8, 10, 11, 19, 31, 33, 34, 46–48, 51–53]
(Figure 4.3, Figure 4.4, Figure 4.5 and
Figure 4.6).
Table .Provisional dosing guidelines for
botulinum toxin B injections for facial rhytids.
BTX-B (Myobloc®)
Muscle site
(units)
No. of injections
Glabella
2,000–3,000
3
Frontalis
1,000–2,500
3–6
Brow lift
300–500 per
side
1 per side
Periorbital
1,000–1,500
per side
1–2 per side

4 Myobloc

(a)
(b)
Figure .Technique of (a) glabella and (b) lateral canthal line injections.
Adverse Events
Complication profiles for BTX-A versus
Myobloc are quite similar with temporary
mild bruising and headaches. Rarely, poten-
tial complications include eyelid and brow
ptosis,
and
asymmetric
brow
elevation
(Figure 4.7). It is imperative to see a fully
qualified and skilled physician with a thor-
ough understanding of injection points and
dosages for optimal results and minimal
complications [43,54,55].
Conclusion
In recent years, there has been a strong surge
in demand from patients for safe and effec-
tive aesthetic solutions to combat signs of
aging manifested by the formation of facial
rhytids in the glabellar, forehead and peri-
orbital regions. The overactive nature of
Figure .Before (a) and after (b) 12 weeks after
treatment with 3,000 U of botulinum toxin type B to
the glabella.
(a)
(b)
Figure .Treatment of frontalis muscle in (a) a male patient and (b) a female patient with 3,000 U of
botulinum toxin type A.


Botulinum Toxins
(a)
(b)
Figure .Before (a) and after (b) 12 weeks after treatment with 3,000 U of botulinum toxin type B to lateral
canthal lines.
the underlying facial muscles has been suc-
cessfully treated by botulinum toxins, which
weaken the muscles and cause paralysis,
and are particularly effective for patients
with prominent hyperfunctional facial lines.
BTX-A and Myobloc are two serotype for-
mulations of botulinum toxin that are avail-
able in the United States. Each has its own
unique mechanism of action, binds serotype-
specific receptors, and targets specific intra-
cellular proteins. Myobloc comes as a ready-
to-use liquid formulation compared to the
BTX-A, which is found in stable powder
form requiring reconstitution before inject-
ing. Although both toxins can elicit an
Figure .Drooping of the left upper eyelid after
botulinum toxin type B injection in the glabella.
Apraclonidine (0.5%) drops, 3 drops to the affected
eye three times a day produces temporary relief of
this problem until it completely resolves.
immune response, further clinical studies are
needed to demonstrate which toxin is more
prone to provoke an immune response. BTX-
A has been used in aesthetic medicine for
several years, with numerous clinical studies
substantiating its efficacy, whereas Myobloc
has only recently been used for aesthetic
indications. Nevertheless, initial findings on
Myobloc indicate that it can deliver more
rapid and greater results in paralysing mus-
cles. The safety profile of Myobloc and BTX-
A is favorable and the likelihood of seri-
ous adverse effects is low; albeit both toxins
are highly potent and extreme care should
be taken, especially when increasing doses
in patients. When there is excess enthu-
siasm from the physician’s part to deliver
formidable aesthetic results by using toxins
vigorously (especially in the lower face), the
side effects become prevalent and trouble-
some. Thus, for a practicing clinician in the
aesthetic field, patient safety through a deep
understanding of best practice and proce-
dure is more paramount to avoid posttreat-
ment complications than securing patient
satisfaction. As studies continue to advance
our understanding of the the mechanism of
action, durability, and safety of Myobloc, the
aesthetic indications and treatment protocols
are bound to expand, capitalizing on its effi-
cacy while minimizing any potential adverse
effects.

4 Myobloc

References
Alster TS, Lupton JR. Botulinum toxin type
B for dynamic glabellar rhytides refractory
to botulinum toxin type A. Dermatol Surg
2003. 29(5):516–518.
Blitzer A, Brin MF, Keen MS, Aviv JE.
Botulinum toxin for the treatment of
hyperfunctional lines of the face. Arch
Otolaryngol Head Neck Surg 1993;119(9):
1018–1022.
Brandt FS, Bellman B. Cosmetic use of
botulinum A exotoxin for the aging neck.
Dermatol Surg 1998;24(11):1232–1234.
Carruthers J, Carruthers A. BOTOX use in
the mid and lower face and neck. Semin
Cutan Med Surg 2001;20(2):85–92.
Carruthers JD, Carruthers JA. Treatment of
glabellar frown lines with C. botulinum-A
exotoxin. J Dermatol Surg Oncol 1992;
18(1):17–21.
Dolimbek BZ, Jankovic J, Atassi MZ. Cross
reaction of tetanus and botulinum
neurotoxins A and B and the boosting
effect of botulinum neurotoxins A and B on
a primary anti-tetanus antibody response.
Immunol Invest 2002;31(3–4):247–262.
Guyuron B, Huddleston SW. Aesthetic
indications for botulinum toxin injection.
Plast Reconstr Surg 1994;93(5):913–918.
Jacob CI. Botulinum neurotoxin type B – a
rapid wrinkle reducer. Semin Cutan Med
Surg 2003;22(2):131–135.
Matarasso A, Matarasso SL, Brandt FS,
Bellman B. Botulinum A exotoxin for the
management of platysma bands. Plast
Reconstr Surg 1999;103(2):645–652;
discussion 653–655.
Sadick NS, Fellows of the American
Academy of Cosmetic Surgeons.
Botulinum toxin type B for glabellar
wrinkles: a prospective open-label response
study. Dermatol Surg 2002;28(9):817–821.
Pierard GE, Lapiere CM. The
microanatomical basis of facial frown lines.
Arch Dermatol 1989;125(8):1090–1092.
Callaway JE, Arezzo JC, Grethlein AJ.
Botulinum toxin type B: an overview of its
biochemistry and preclinical
pharmacology. Semin Cutan Med Surg
2001;20(2):127–136.
Dressler D, Bigalke H. Botulinum toxin
type B de novo therapy of cervical dystonia:
frequency of antibody induced therapy
failure. J Neurol 2005;252(8):904–907.
Han Y, Stevens AL, Dashtipour K, et al. A
mixed treatment comparison to compare
the efficacy and safety of botulinum toxin
treatments for cervical dystonia. J Neurol
2016;263(4):772–780.
Hosp C, Naumann MK, Hamm H.
Botulinum toxin treatment of autonomic
disorders: focal hyperhidrosis and
sialorrhea. Semin Neurol 2016;36(1):20–28.
Aoki KR. Pharmacology and immunology
of botulinum neurotoxins. Int Ophthalmol
Clin 2005;45(3):25–37.
Callaway JE, Arezzo JC, Grethlein AJ.
Botulinum toxin type B: an overview of its
biochemistry and preclinical
pharmacology. Dis Mon 2002;48(5):
367–383.
Dong M, Tepp WH, Liu H, Johnson EA,
et al. Mechanism of botulinum neurotoxin
B and G entry into hippocampal neurons.
J Cell Biol 2007;179(7):1511–1522.
Flynn TC, Clark RE 2nd. Botulinum toxin
type B (MYOBLOC) versus botulinum
toxin type A (BOTOX) frontalis study: rate
of onset and radius of diffusion. Dermatol
Surg 2003;29(5):519–522; discussion 522.
Oguma K, Fujinaga Y, Inoue K. Structure
and function of Clostridium botulinum
toxins. Microbiol Immunol 1995;39(3):
161–168.
Setler P. The biochemistry of botulinum
toxin type B. Neurology 2000;55(12 Suppl
5):S22–S28.
Simpson LL. Molecular pharmacology of
botulinum toxin and tetanus toxin. Annu
Rev Pharmacol Toxicol 1986;26:427–453.
Atassi MZ, Dolimbek BZ, Jankovic J, et al.
Molecular recognition of botulinum
neurotoxin B heavy chain by human


Botulinum Toxins
antibodies from cervical dystonia patients
that develop immunoresistance to toxin
treatment. Mol Immunol 2008;45(15):
3878–3888.
Berman B, Seeberger L, Kumar R.
Long-term safety, efficacy, dosing, and
development of resistance with botulinum
toxin type B in cervical dystonia. Mov
Disord 2005;20(2):233–237.
Jankovic J, Hunter C, Dolimbek BZ, et al.
Clinico-immunologic aspects of botulinum
toxin type B treatment of cervical dystonia.
Neurology 2006;67(12):2233–2235.
Callaway JE. Botulinum toxin type B
(Myobloc): pharmacology and
biochemistry. Clin Dermatol 2004;22(1):
23–28.
Carruthers A, Carruthers J, Flynn TC,
Leong MS. Dose-finding, safety, and
tolerability study of botulinum toxin type B
for the treatment of hyperfunctional
glabellar lines. Dermatol Surg 2007;33(1
Spec No.):S60–S68.
Gilsdorf J, Gul N, Smith LA. Expression,
purification, and characterization of
Clostridium botulinum type B light
chain. Protein Expr Purif 2006;46(2):256–
267.
Kranz G, Paul A, Voller B, et al. Long-term
efficacy and respective potencies of
botulinum toxin A and B: a randomized,
double-blind study. Br J Dermatol
2011;164(1):176–181.
Sadick NS. Prospective open-label study of
botulinum toxin type B (Myobloc) at doses
of 2,400 and 3,000 U for the treatment of
glabellar wrinkles. Dermatol Surg 2003;
29(5):501–507; discussion 507.
Ramirez AL, Reeck J, Maas CS. Botulinum
toxin type B (MyoBloc) in the management
of hyperkinetic facial lines. Otolaryngol
Head Neck Surg 2002;126(5):459–467.
Lowe PL, Lowe NJ. Botulinum toxin type B:
pH change reduces injection pain, retains
efficacy. Dermatol Surg 2014;40(12):
1328–1333.
Lowe NJ, Yamauchi PS, Lask GP, et al.
Botulinum toxins types A and B for brow
furrows: preliminary experiences with type
B toxin dosing. J Cosmet Laser Ther
2002;4(1):15–18.
Kim EJ, Ramirez AL Reeck JB, Maas CS.
The role of botulinum toxin type B
(Myobloc) in the treatment of hyperkinetic
facial lines. Plast Reconstr Surg 2003;112
(5 Suppl):88S–93S; discussion 94S–97S.
Sadick NS, Herman AR. Comparison of
botulinum toxins A and B in the aesthetic
treatment of facial rhytides. Dermatol Surg
2003;29(4):340–347.
Patel S, Martino D. Cervical dystonia: from
pathophysiology to pharmacotherapy.
Behav Neurol 2013;26(4):275–282.
Pappert EJ, Germanson T, Myobloc/
Neurobloc European Cervical Dystonia
Study. Botulinum toxin type B vs. type A in
toxin-naive patients with cervical dystonia:
Randomized, double-blind, noninferiority
trial. Mov Disord 2008;23(4):510–17.
Chapman MA, Barron R, Tanis DC, et al.,
Comparison of botulinum neurotoxin
preparations for the treatment of
cervical dystonia. Clin Ther 2007;29(7):
1325–1337.
Ascher B, Zakine B, Kestemont P,
Baspeyras M, et al. A multicenter,
randomized, double-blind, placebo-
controlled study of efficacy and safety of 3
doses of botulinum toxin A in the
treatment of glabellar lines. J Am Acad
Dermatol 2004;51(2):223–233.
Baumann L, Slezinger A, Vujevich J, Halem
M, et al. A double-blinded, randomized,
placebo-controlled pilot study of the safety
and efficacy of Myobloc (botulinum toxin
type B)-purified neurotoxin complex for
the treatment of crow’s feet: a
double-blinded, placebo-controlled trial.
Dermatol Surg 2003;29(5):508–515.
Blitzer A, Binder WJ, Aviv JE, et al. The
management of hyperfunctional facial lines
with botulinum toxin. A collaborative study
of 210 injection sites in 162 patients. Arch
Otolaryngol Head Neck Surg 1997;123(4):
389–392.
Carruthers JA, Lowe NJ, Menter MA, et al.
A multicenter, double-blind, randomized,
placebo-controlled study of the efficacy and

4 Myobloc

safety of botulinum toxin type A in the
treatment of glabellar lines. J Am Acad
Dermatol 2002;46(6):840–849.
Erickson BP, Lee WW, Cohen J,
Grunebaum LD. The role of neurotoxins in
the periorbital and midfacial areas. Facial
Plast Surg Clin North Am 2015;23(2):
243–255.
Fagien S, Brandt FS. Primary and
adjunctive use of botulinum toxin type A
(Botox) in facial aesthetic surgery: beyond
the glabella. Clin Plast Surg 2001;28(1):
127–148.
Flynn TC, Carruthers JA, Carruthers JA.
Botulinum-A toxin treatment of the lower
eyelid improves infraorbital rhytides and
widens the eye. Dermatol Surg 2001;27(8):
703–708.
Foster JA, Barnhorst D, Papay F, et al. The
use of botulinum A toxin to ameliorate
facial kinetic frown lines. Ophthalmology
1996;103(4):618–622.
Hankins CL, Strimling R, Rogers GS.
Botulinum A toxin for glabellar wrinkles.
Dose and response. Dermatol Surg
1998;24(11):1181–1183.
Huilgol SC, Carruthers A, Carruthers JD.
Raising eyebrows with botulinum toxin.
Dermatol Surg 1999;25(5):373–375;
discussion 376.
Lew MF, Adornato BT, Duane DD, et al.
Botulinum toxin type B: a double-blind,
placebo-controlled, safety and efficacy
study in cervical dystonia. Neurology
1997;49(3):701–707.
Lowe NJ, Maxwell A, Harper H. Botulinum
A exotoxin for glabellar folds: a
double-blind, placebo-controlled study
with an electromyographic injection
technique. J Am Acad Dermatol 1996;35(4):
569–572.
Spencer JM, Gordon M, Goldberg DJ.
Botulinum B treatment of the glabellar
and frontalis regions: a dose response
analysis. J Cosmet Laser Ther 2002;4(1):
19–23.
Vecchione TR. Glabellar frown lines: direct
excision, an evaluation of the scars. Plast
Reconstr Surg 1990;86(1):46–52.
Knize DM. Muscles that act on glabellar
skin: a closer look. Plast Reconstr Surg
2000;105(1):350–361.
Dubow J, Kim A, Leikin J, et al. Visual
system side effects caused by
parasympathetic dysfunction after
botulinum toxin type B injections. Mov
Disord 2005;20(7):877–880.
Francisco GE. Botulinum toxin: dosing and
dilution. Am J Phys Med Rehabil
2004;83(10 Suppl):S30–S37.



Abobotulinumtoxin: Development and Aesthetic Usage
Gary D. Monheit, MD
Total Skin and Beauty Dermatology Center, PC Private Practice,
Associate Clinical Professor, Department of Dermatology, Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, USA
Clinical studies
Since
the
muscle-paralyzing
activity
of
botulinum toxin (BoNT) was first noted in
the nineteenth century, the clinical usefulness
of this “most poisonous of all poisons” [1] has
evolved into a diversity of therapeutic uses.
Currently, several commercial preparations
of botulinum toxin serotype A (BoNTA) are
available for the treatment of strabismus,
blepharospasm, cervical dystonia, migraine,
laryngeal spasms, and other disorders of
muscle contraction. Since Carruthers and
Carruthers first published their sentinel arti-
cle on the relationship of muscle activity with
wrinkles and its correlation with BoNTA, the
aesthetic uses have expanded greatly [2].
Onabotulinumtoxin
(Botox®,
Allergan,
Irvine, California) was first FDA approved
in 2002 for glabellar lines, but since then
it has been applied off-label for other
facial dynamic wrinkles and other aesthetic
indications [3].
Abobotulinumtoxin
(Dysport®;
Ipsen,
Medicis
US
and
Azzalure®,
Galderma,
Europe) was first licensed for medical usage
in Europe in 1990[4]. Clinical studies for
aesthetic indications were first performed
in Europe 2002 and 2003, followed by US
FDA studies until 2009. FDA approval for
treatment of glabellar frown lines occurred
May 2009.
All
the
preparations
are
BONTA
molecules, but there are some technical
differences in the formulations. Though
most clinical properties seem to act the
same, there may be some subtle differences
the clinician must understand. It is thus
important to review the science of the
molecule, its formulations, and its physio-
logic action to use each of the preparations
correctly.
To use this new product correctly it is nec-
essary to understand the following points:
1. The science of the BoNTA molecule and
its differences.
2. The clinical studies demonstrating efficacy
and safety.
3. Differences in the products.
4. Experience in worldwide usage.
The three most widely used products –
obobotulinumtoxin
(Botox®),
abobo-
tulinumtoxin
(Dysport®/Azzalure®)
and
incobotulinumtoxin (Xeomin®) – are pro-
duced from different strains of bacteria,
purified using different methods, and there-
fore they have distinct properties; however,
the toxins are all derived from the BoNTA
molecule, which is a polypeptide with a light
and a heavy chain activated by “nicking’
at the junction of the disulfide bond. The
BoNTA
cleaves
SNAP-25
(synaptosomal
associated protein, 25 kDa), blocking the
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:


Botulinum Toxins
neuromuscular junctions, thus interrupting
neurotransmission of acetylcholine [5].
The
units
of
Azzalure/Dysport
and
Vistable/Botox are not interchangeable, as
different bioassays were used for measuring
their activities [6, 7]. Azzalure and Dysport
are quantified in Speywood Units (s.U.)
and are therefore collectively referred to as
BoNTA (Speywood Unit) [8]. It is important
to understand this concept as the assays
used to compare these products unit by unit
are not reliable, thus putting question into
most direct comparisons of other products.
This includes efficacy and safety as these are
directly related to dosage and dilution.
The BoNTA (Speywood unit) toxin exists
as a complex with a surrounding coat of
protective proteins: the hemagglutinin and
nontoxin nonhemagglutinin proteins. The
surrounding complex protein can serve to
protect the neurotoxin from its potential
destruction by the low pH of stomach acid.
On absorption as the pH becomes physi-
ologic, the outer protein coat releases the
neurotoxin [9]. Similarly, the full complex
found in the vial prior to injection (750 or
900 kDa) dissociates upon injection to the
naked 150 kDa neurotoxin molecule [10].
Though both the Botox and Dysport
neurotoxin molecules are the same weight
(150 kDa), the complexes are different sizes
due to variations in production. Botox
is 900 kDa and Dysport is approximately
750 kDa [11]. The influence of the complexes
once injected is unknown, especially since
dissociation occurs at physiologic pH. Ques-
tions concerning field of effect, diffusion,
and onset of action of each toxin are not
fully answered. We do know that each has
different units of potency (Botox units versus
Dysport units) and this has a major impact
on its clinical significance.
Technical differences in the two products
are less than the similarities. The complex
size is as follows:
1. OnabotulinumtoxinA: 900 kDa
2. AbobotulinumtoxinA: 500–700 kDa
Thus, the relevance of the accessory pro-
teins as well as the molecular weight is under
question [10]. Other differences are in the
composition of the product, which is summa-
rized in Table 5.1. Protein content is slightly
greater in onabotulinumtoxinA with more
serum albumin, but the carriers – NaCl and
lactose – do differ; it is unknown if this
makes a clinical difference. Note that dissoci-
ation of the 150 kDa molecule from its acces-
sory proteins does occur rapidly, in the vial
Table .Constituents of botulinum toxin.
Dysport []
Botox cosmetic []
Vial size
300 U
100 Ua
Composition
Clostridium botulinum toxin
type A hemagglutinin complex
Clostridium botulinum toxin type A
hemagglutinin complex
125 μg (0.125 mg) human
serum albumin
500 μg (0.5 mg) human serum
albumin
2.5 mg lactose
0.9 mg sodium chloride
Molecular weight
(neurotoxin)
150 kDa
150 kDa
Bulk active substance
(total protein content)
∼3 ng
∼5 ng
Storage
(post-reconstitution)
2–8◦C/until vial expiration
(2–8◦C / use within 4 h)
2–8◦C / 36 months
(2–8◦C / use within 4 h)
aBOTOX Cosmetic is also available in a 50 U vial. The 100 U vial is referenced in this comparison.

5 Abobotulinumtoxin: Development and Aesthetic Usage

with reconstitution or in the needle prior to
injection, but definitely upon injection when
pH returns to physiologic status. Eisele has
shown that this takes place in less than a
minute [12]. This gives further evidence that
molecular weights of the different toxins do
not influence their behavior in vivo. The argu-
ment that spread or diffusion is based on
molecular weight of the toxin is thus not
relevant.
Clinical Studies Performed for
Abobotulinumtoxin
Aesthetic
studies
for
abobotulinumtoxin
(Dysport) followed a similar pattern as those
performed for Botox by Carruthers and
Carruthers a few years earlier. The first Euro-
pean studies for aesthetic usage of Dysport
were performed by Ascher in the later 1990s
and again by Ascher and Rzany in 2004 and
2006 [13]. A “dose-finding” study was first
performed on 119 patients using placebo,
25, 50 and 75 U in a double-blinded control.
The subjects were measured for efficacy as a
responder at 1 month and then assessed for
safety and duration. A responder rate of over
80% was found for all three groups, and at
6 months two-thirds of the treated patients
were still responders. There was a favorable
safety profile of 7% mild adverse events, with
headache the most common. There were
no reported cases of blepharoptosis or a
diplopia. The result suggested 50 Speywood
units was the optimal dosage for the glabella
with 10 units injected into each of five
glabellar sites [14].
This was followed by the US studies
beginning in 2003 and extending to the
present. The Inamed, Ipsen and Medicis
studies included phase II, phase III single
and repeat dosage studies with a total of
2,300 patients assessed for efficacy and safety.
All studies evaluated efficacy and safety of
glabellar frown lines with rating scales at
rest and at maximal frown. The US stud-
ies used the same injection points as the US
onabotulinumtoxinA trials and were evalu-
ated at maximal frown as its end points. In
Europe, end points for response were deter-
mined at rest for efficacy and the lateral cor-
rugator injection points were placed 0.5 cm
more medial; however, the results for efficacy
and safety for both studies were similar.
Phase II trials involved a dosage-ranging
study including placebo, 20, 50 and 75 U.
There was a 90% responder rate at both 50 U
and 75 U. All doses were well tolerated with
only minor side effects including headache,
needle pricks, and bruising. Blepharoptosis
was observed in only three patients, of which
only one demonstrated to the investigator a
true clinical ptosis. Ptosis had been reported
in other product studies but not in other stud-
ies involving abobotulinumtoxinA [15].
Antibody production has always been
of concern with clinical usage of BoNTA,
but has only been studied with cervical
dystonia [16]. None of the patients in this
study showed any evidence of neutralizing
antibodies either at baseline or on follow-up
evaluations. From these observations, the
50 U dosage was recommended as the opti-
mal dose for safety and efficacy. The lower
dosage of 50 U was chosen for all of the
phase III trials [17].
The
initial
single-dosage,
placebo-
controlled,
double-blinded,
randomized
trial of glabellar frown lines for FDA Phase
III included over 400 subjects followed
for 150 days [18]. The patients were then
enrolled in a repeat dosage trial for 23 months
including four cycles of repeat dosage when
the patient’s frown lines returned to baseline.
The patients were evaluated for efficacy
(i.e., number of responders at 30 days and
duration of response) and safety. Over 90%
responder rate was recorded at 30 days,
with duration at 4 months of 40% respon-
ders and at 5 months 25% were still full
responders [19]. This produced a very similar
efficacy case for responders and duration as
onabotulinumtoxinA.
One factor reported by the subjects was
an onset of action within 1–2 days. Subse-
quent studies have included a diary that the
patient recorded when an onset of effect was
first noted. In three of the studies the onset
was recorded: 50% of subjects noted an onset


Botulinum Toxins
within 2 days and 80% noted it within 3
days [20]. Onset studies were to follow with
onabotulinumtoxinA and incobotulinumtox-
inA toxins which were performed in a simi-
lar manner and with results that were com-
parable. Re-injection treatment studies were
performed to ensure that repeated exposure
to the toxin did not influence efficacy or
duration.
The repeat administration studies involved
768 individuals from phase III clinical tri-
als who received up to six repeated treat-
ments over 17 months. A patient was rein-
jected when glabellar wrinkle lines returned
to baseline. The patients were followed for
efficacy and safety including adverse effects
and assessment of serum-neutralizing anti-
bodies to abobotulinumtoxinA. Results con-
firmed continued effectiveness throughout
the study with no increase of active events
and no patients developed neutralizing anti-
bodies.
The possibility of blocking antibodies influ-
encing efficacy and toxin action has been
noted with large therapeutic doses given over
years for treatment of spastic torticolis and
cerebral palsy. Antibody blocking effect, as of
this time, has not been recorded with the cos-
metic use of abobotulinumtoxinA [21].
As the data of these initial trials were
reviewed, it was noted that efficacy and dura-
tion of action for men was less than that
for women on a 50 U dosage for treating
the glabella. This observation stimulated a
variable-dose study which stratified patients
by race/ethnicity, sex, and randomized by
muscle mass. The muscle mass groups were
to receive a single treatment of various doses
of abobotulinumtoxinA. Abobotulinumtox-
inA was administered as a single dose of
50, 60 or 70 U for women and 60, 70, or
80 U for men based on glabellar muscle mass.
This is based on procerus/corrugator muscle
mass, small, medium or large. This was deter-
mined by the observation of an active frown,
noted individual muscle bulging, length of the
infrabrow space, and brow depression. Effi-
cacy and duration was evaluated during a 5
month period. The results indicated that 87%
of men and women had full efficacy at 30 days
with a mean duration of 109 days for both
men and women with no difference in eth-
nicity or gender [22]. Duration of action was
found to be increased with the greater doses
for larger muscle mass.
Though
clinicians
have
individualized
abobotulinumtoxinA glabellar treatment for
sex, ethnicity, and muscle mass in clinical
practice, this was the first controlled clinical
study to verify this common practice.
Dysport was first licensed for medical
usage in Europe in 1990. Clinical studies for
aesthetic indications were first performed in
Europe in 2002 and 2003, followed by US
FDA studies in 2009. FDA approval for treat-
ment of glabellar frown lines occurred May
2009. Table 5.2 summarizes the differences
between the two formulations of BoNTA.
While variations in progenitor complex size
as described earlier may exist and the excipi-
ents differ, it must be stressed that the “active”
ingredient is thought to be identical and as
such the clinical properties of the two agents
are largely similar. Purported variations in
clinical behavior are likely to be subtle when
controlled for volume, toxin concentration,
and injection technique.
The most important difference between
onabotulinumtoxinA and abobotulinumtox-
inA is in the activity units employed by their
respective manufacturers: Botox units (b.U.)
for onabotulinumtoxinA and Speywood units
(s.U.) for abobotulinumtoxinA. Both define
one unit as the quantity necessary to kill 50%
of mice (LD50) with an intraperitoneal injec-
tion. Because of differences in the experimen-
tal design of their murine assays, however, the
units are not equivalent. The Dysport assay is
undeniably more sensitive, that is, less toxin
is required to kill a mouse when toxin of any
formulation is tested in this assay versus the
Botox assay [23]. Indeed, in a small study it
was shown that, when tested in the Dysport
assay, the LD50 of Botox is achieved with
0.32 b.U. (68% less product than that required
for LD50 in the Botox assay) [23]. Therefore,
a Speywood unit corresponds to a smaller
quantity of active toxin than does a Botox

5 Abobotulinumtoxin: Development and Aesthetic Usage

Table .Overview of product composition for the FDA approved Botulinum toxin serotype A agents.
IncobotulinumtoxinA (Xeomin) is only approved for therapeutic use [35–40].
Product
Botox
Dysport
Xeomin
Manufacturer
Ipsen (Europe)
Medicis (USA)
Allergan
Merz Pharmaceuticals
Units per vial
100 b.U.
300 s.U.
(for cosmetic use)
50 or 100 U
Active ingredient
(molecular weight)
Botulinum toxin
serotype A complex
(900 kDa)
Botulinum toxin
serotype A complex
(500–900 kDa)a
Uncomplexed
Botulinum toxin
serotype A (150 kDa)
Total toxin protein per
vial (active toxin +
NAPsb)
5 ng
2.61 ng
0.6 ng (in 100 U)
Excipients
Human serum
albumin 500 μg
NaCl 0.9 mg
Human serum
albumin 125 μg
Lactose 2.5 mg
Human serum
albumin 1 mg
Sucrose 4.7 mg
Bacterial source
Clostridium
botulinum,
Hall strainc
Clostridium
botulinum,
Hall strainc
Clostridium
botulinum,
Hall strainc
Storage conditions
2–8◦C
2–8◦C
Up to 25◦C
Purification process
Dialysis and acid
precipitation then
vacuum dried
Column
chromatography then
freeze-dried
(lyophilized)
Column
chromatography then
freeze-dried
(lyophilized)
aMolecular weight of AboA is not firmly established –See discussion in text.
bNeurotoxin-associated proteins
cThere are numerous “Hall strains” and the manufacturers do not necessarily use identical bacteria.
unit. The exact potency ratio between s.U.
and b.U. remains an open question. Since, in
any competitive marketplace, product inter-
changeability is not a desirable attribute,
the BoNTA manufacturers have predictably
emphasized the uniqueness of their formula-
tions and have discouraged the use of any unit
conversion factors. Nevertheless, practition-
ers have sought to define a conversion factor
to guide the novice injector when transition-
ing from one toxin to the other for a given
application.
Numerous in vivo and in vitro studies have
attempted to define this conversion factor
with conflicting results. For a comprehensive
review of this literature, please refer to the
recently published review by Karsai et al.
[24]. Only a brief summary of the pertinent
human studies follows. A 2004 meta-analysis,
using
Cochrane
review
methodology,
identified
four
high-quality
comparative
clinical studies all examining neurologic indi-
cations, two employed a 1 : 4 (OnaA:AboA)
dose ratio, one a 1 : 3 ratio, and the last
used 1 : 3 and 1 : 4 in separate arms [25].
This analysis concluded that a 1 : 4 ratio
was too high, and a 1 : 3 ratio approached
bioequivalence although the included studies
suggested that an even lower ratio might
be more appropriate. An independently
funded,
double-blind
study
of
Dysport
versus Botox for the treatment of glabellar
lines found a longer duration of action as
assessed by electromyographic studies with
Dysport used at a 1 : 3 ratio. This led the
authors to conclude that the bioequivalent
ratio was less than 1 : 3 [26]. Lowe et al.
examined the relative effects of a 1 : 2.5
dose ratio on glabellar lines assessed by
blinded investigator rating and found greater


Botulinum Toxins
longevity with Botox [27]. Therefore, while
the
preponderance
of
current
evidence
supports a dose ratio of no more than 1 : 3,
a more precise definition awaits additional
controlled, head-to-head comparisons with
ideally objective measurements of muscle
activity. The authors recommend a con-
version factor of 1 : 2.5, which has become
the most commonly quoted unit dose ratio
among experienced injectors. The multiple
studies that underpinned the FDA approved
dosages for glabellar lines (50 s.U. of Dys-
port and 20 b.U. of Botox) demonstrated
comparable efficacy with the two BoNTA
products, further supporting the 1 : 2.5 ratio
as a starting point for cosmetic applications.
The perception that an increased capacity
for a toxin to diffuse from the site of injec-
tion translates into increased side effects has
encouraged a lively debate among the manu-
facturers of the BoNTA products. The ques-
tion of diffusion has mostly been investigated
in humans by measuring anhidrotic haloes
after injecting equal volumes of each agent
into the forehead. Some comparative stud-
ies of Botox and Dysport have demonstrated
that anhidrotic haloes are significantly larger
for Dysport [28, 29]. The only double-blind
randomized study utilized a Botox : Dysport
unit ratio of 1 : 3, and one could argue that
the increased anhidrotic haloes observed in
this study with Dysport is a consequence of
not using equipotent dosages. In other words,
diffusion is primarily driven by concentra-
tion gradients, and it would be expected that
a higher injected concentration of neuro-
toxin would result in greater diffusion. In an
unblinded study, Trindade De Almeida et al.
[29] examined three different dose ratios:
1 : 2.5, 1: 3, and 1 : 4. They found significantly
increased anhidrotic haloes with all dose
ratios (the mean absolute increase in area
of the anhidrotic halo was 1.2 cm2 with the
lowest Dysport dosage). These results were
challenged by a similarly designed compar-
ative trial conducted by Hexsel et al. which
found no significant difference in the field
of anhidrosis using only the more widely
accepted equipotent dose ratio of 1 : 2.5 [30].
Hexsel et al. reported taking great care to
standardize injection technique, which would
certainly influence results. Of note, the first
two described studies [28,29] were sponsored
by Allergan and the third [30] by Ipsen. A
definitive answer to this question will await
an impartial, double-blind study comparing
truly equipotent injections of neurotoxins.
In conclusion, it must be emphasized that
subtle differences in the properties of the
BoNTA formulations may exist. Extant data
on product composition, diffusion proper-
ties, and relative clinical potencies remains
inconclusive. Indeed, the number and types
of NAPs probably differ between BoNTA
products. NAPs have known biologic rel-
evance in the pathogenesis of food-borne
botulism [31]. One recent study demon-
strated that a hemagglutinin protein binds E-
cadherin to facilitate passage of toxin through
epithelial tight junctions within the alimen-
tary canal [32]. This raises the unexplored
and previously discounted possibility that
NAPs could have other specific biologic func-
tions, some of which might impinge on the
neuromuscular activity of injected BoNTA.
Until we possess a better understanding of
these various issues, the injector is advised
to think and treat independently with each
BoNTA product – as one learns a foreign lan-
guage – and avoid converting for usage.
AbobotulinumtoxinA
has
been
used
worldwide for aesthetic needs for over 10
years. Those clinicians who have the greatest
experience have defined injection points,
minimal dosage, and a range of doses for
each of the facial areas.
Both glabella and crow’s feet have been
well studied by Asher with results compa-
rable to onabotulinumtoxinA [33]. Forehead,
eyebrows and full upper face are successfully
treated with abobotulinumtoxinA in a similar
fashion to onabotulinumtoxinA.
AbobotulinumtoxinA has been used in
clinical cosmetic practice worldwide over the
past decade with similar injection points
and techniques as onabotulinumtoxinA, but
with different dosages. A full understanding
of anatomy and product effect on distinc-
tive facial muscles is essential for ensur-
ing optimal treatment results and should be

5 Abobotulinumtoxin: Development and Aesthetic Usage

International Board Botulinum Toxin
January 12–13, 2009
Paris, France
1. Benjamin Ascher: Chairman – France
2. Sergio Talarico: Co-Chairman – Brazil
3. Daniel Cassuto: Italy
4. Sergio Escobar: Argentina
5. Doris Hexsel: Brazil
6. Pedro Jaen Olasolo: Spain
7. Gary Monheit: USA
8. Berthold Rzany: Germany
9. Maurizio Viel: London
10. Alexandre Kaoukhov – France/Galderma
Research
Sponsoring: Galderma
Ipsen
acquired through proper training. Since there
are only a few clinical studies concerning off-
label indications, one of the original consen-
sus conferences was held in Paris in January,
2009, which provided general guidelines for
effective and safe use of abobotulinumtox-
inA on the generally useful yet off-label sites
for injection. This focused on the European
experience and served as one of the initial
guides for therapy in the United States. These
have included the common upper face sites –
the glabella, forehead, brow, crows’ feet, and
eyelid – and other less common facial sites
including bunny lines, depressor angularis
oris, obicularis oris, mentalis, and platysma.
These recommendations gave guidelines to
starting dosage and range for each site as well
as injection points and technique.
On January 12–13, 2009, an International
Board of Botulinum Toxin was assembled in
Paris to develop a consensus of dosage rec-
ommendation for the use of abobotulinum-
toxin. The following physicians were part of
the board giving a consensus of recommen-
dations for common treatment sites.
The site-specific recommendations were
given as shown in Table 5.3.
Treatment of the Upper Face
Glabellar lines
Injection of the glabella is the original and
by far most common cosmetic usage of
BoNTA. Numerous randomized, placebo-
controlled
trials
have
demonstrated
the
efficacy of
abobotulinumtoxinA
for
this
indication.
The standard five-injection point approach
is appropriate for most patients with two
Table .Recommended Dysport® dosage for upper face.
Total usual dose
Dose range
Indications
(Dysport® units)
(Dysport® units)
Glabella
50
30–70
Forehead
40–50
40–70
Crows’ feet
30 × 2
20–50 × 2
Lateral eyebrow lift
20 × 2
20–40 × 2
Glabella & forehead
90–100
70–140
Glabella & lateral eyebrow lift
90
50–110
Complete upper third face
150
110–240


Botulinum Toxins
Figure .Injection sites for glabellar line treatment.
Blue dots, corrugator insertion/orbicularis oculi
fibers; yellow dots, corrugator body; green dot,
procerus.
injections in each corrugator and one injec-
tion in the procerus as shown in Figure 5.1.
The procerus and medial corrugators are
injected deeply and directly into the bodies
of the muscles, which are easily identified at
maximal frown in most patients. The lateral
corrugator is injected slightly more superfi-
cially where it inserts into the dermis and
medial to the midpupillary line. These lat-
eral injections also target fibers of the orbic-
ularis oculi. All injections should be 1.0 cm
(approximately one finger’s breadth) above
the orbital rim to limit the risk of eyelid pto-
sis from the spread of toxin to the levator
palpebrae muscle. The measurement should
be made from the orbital rim and from the
brow itself as a dropped brow may mislead
the physician to inject the toxin below the
orbital rim. This can cause toxin penetra-
tion through the orbital septum and weaken
the levator muscle. Injecting too high on the
forehead – above 1 cm at the lateral glabel-
lar site – can also cause ptosis in those indi-
viduals who recruit eyelid elevation from the
frontalis muscle.
The on-label total dose of abobotulinum-
toxinA is 50 s.U., divided evenly among the
five injections, in women and those with
small to medium muscle mass with moder-
ate frown. As always, doses must be adjusted
for the strength of individual muscles and the
patient’s desired outcome. The authors do use
a higher dose of toxin in men and may add
two additional injection points at the mid-
pupillary line in patients with bulky corruga-
tors; however, we rarely exceed a total dose of
80 s.U. in any patient.
In the initial clinical trials with abobo-
tulinumtoxinA, men did not have as robust
a response to the 50 s.U. dose as did women,
presumably due to increased muscle mass.
This inspired a further study which varied
abobotulinumtoxinA dosage with corruga-
tor/procerus volume. Men with mild frown
(small muscle mass) received 60 s.U., moder-
ate 70 s.U., and severe 80 s.U. Correlating the
dose with muscle mass raised the response
rate of men to that of women and increased
the longevity of response in all subjects [34].
Forehead Lines
Although an off-label usage, BoNTA provides
excellent smoothing of forehead rhytides and
is, in general, a very gratifying procedure for
patients when performed properly. However,
the more severe forms of forehead wrinkles
cannot be corrected by BoNTA denervation
alone and may need soft tissue augmentation.
The goal for the treatment of forehead
wrinkles is to soften the undesirable lines
without causing brow ptosis or eliminating all
expressiveness on the upper face. A conser-
vative approach is preferred, informing the
patient preoperatively that more than one
treatment may be needed to reach the desired
level of wrinkle reduction while avoiding
undesirable side effects. The patient is asked
to forcefully raise his/her eyebrows and the
strength of the frontalis is assessed. Any dis-
crepancy in brow position at baseline and at
maximal contraction is noted and brought to
the attention of the patient. It is especially
important to note brow asymmetry, photo-
graph it and bring it to the patient’s attention
prior to treatment. Using appropriate doses,
it can be corrected by using appropriate fore-
head dosage and injection points. A compen-
satory downward dose adjustment should be
made on the side with the “higher” eyebrow.

5 Abobotulinumtoxin: Development and Aesthetic Usage

Figure .Forehead line treatment in women.
Variation favored by authors to accentuate the
lateral arching of the feminine brow pattern.
In the “average” brow, 5–10 s.U. are injected
in four to six sites at least 2.5–3.0 cm above
the orbital rim. Administration more inferi-
orly greatly increases the risk of brow ptosis
as can be seen with onabotulinumtoxin.
The authors commonly use a V-shaped
configuration for injections in women (Fig-
ure 5.2). While often desirable in females,
this should be avoided in male patients. This
approach can also produce an excessively
arched brow (the mephisto sign or “Dr. Spock
look”). This is prevented with a high lat-
eral forehead injection above the tail of the
brow. If an excessive arched brow occurs, it
can be corrected with a small dose of addi-
tional toxin 1–2 cm superior to the apex of the
arched brow.
Unless no significant glabellar lines are
present, the authors commonly inject the
glabella simultaneously whenever treating
forehead wrinkles. This generally produces
better overall aesthetic results and the con-
comitant paralysis of the brow depressors
reduces the incidence of brow ptosis.
Lateral Eyebrow Lift
The paresis of muscles of facial expression
not only smooths dynamic wrinkles but can
also influence the resting position of various
facial elements. This property has been suc-
cessfully exploited to lift the eyebrow, cor-
recting mild brow ptosis, restoring a youthful
brow arch, and giving the eye a more “open”
appearance.
The vertical fibers of the lateral orbicularis
oculi function to depress the lateral brow. For
this chemical brow lift, the authors typically
inject 5 U of onabotulinumtoxinA or 10 U of
abobotulinumtoxinA intradermally at the lat-
eral tail of the eyebrow 5–7 mm superolat-
eral to the orbital rim (Figure 5.3a). If per-
formed only for brow lift and not to correct
frown lines, an additional injection into each
(a)
(b)
Figure .The chemical brow lift: (a) Injection sites for a chemical brow lift if performed alone are shown (5–10
s.U. or 3–5 b.U. per injection site). This patient had a male pattern eyebrow without perceptible arching. She
received abobotulinumtoxinA treatment to the lateral tail of each eyebrow (10 s.U. per side), into the
corrugators bodies (total 20 s.U.), and to the frontalis (20 s.U.). (b) Posttreatment photo at 33 days reveals an
elevated and laterally arched feminine brow.


Botulinum Toxins
(a)
(b)
Figure .Crow’s feet injection. (a) Standard three point crow’s feet injection. (b) This patient received 5 b.U. at
each point and had an excellent response.
corrugator body is typically added to com-
plete the chemical brow lift. More com-
monly, the lateral brow lift injection is car-
ried out in tandem with other upper face
treatments, especially injection of the glabel-
lar complex and forehead. In combination
with medial and central frontalis denerva-
tion, which tends to lower the medial brow, a
pleasingly arched female pattern eyebrow can
be shaped (Figure 5.3b).
Crow’s Feet
Lateral perioribital wrinkling is one of the
earliest signs of aging. Hyperkinetic lateral
canthal lines are effectively treated with neu-
rotoxin.
Crow’s feet are typically treated with three
equal injections of 10–15 s.U. evenly spaced
along an arc lying at least 1 cm external to
the orbital rim to avoid diffusion to the palpe-
bral portion of the orbicularis oculi or to the
levator palpebrae muscle (Figure 5.4a,b). The
middle injection is placed in line with the
lateral canthus. Injections flanking this point
at 8–10 mm are then placed, but their exact
positioning depends on the width of the indi-
vidual’s canthal lines. The highest crow’s feet
injection is inferior to the lateral eyebrow
tail injection described earlier for a chemical
brow lift.
An additional injection of 2 s.U. microin-
jections can be judiciously placed in the lower
lid at the midpupillary line 2 mm below the
tarsal plate (Figure 5.5). This will flatten the
bulging muscle and create an image of an
“open eye.” Overaggressive treatment may,
though, create an unwanted ectropion.
Treatment of the Lower Face
Treatment of the lower face should be per-
formed with more precaution as overdosage
leads to significant dysfunction of the obic-
ularis oris, creating an asymmetric smile
and oral aperture. I would also recommend
the more concentrated dilution of 1.5 cm3
saline per 300 U vial to titrate, the toxin for
Figure .Location of lower palpebral injection. A
tiny dose of toxin into the lower eyelid can create a
more youthful “open”eye (2 s.U. or 0.5–1 b.U.).

5 Abobotulinumtoxin: Development and Aesthetic Usage

Table .Recommended Dysport® dosage for lower face.
Indications
Total usual dose (Dysport® units)
Dose range (Dysport® units)
Obicularis oris
2.5 per injection point
10–15 total
Depressor angularis oris
5–10 per site
10–20
Mentalis
5–10 per site
10–20
Platysma
5–10 per injection point
20–40
Maximum total dosage 50/side
contouring within appropriated muscles.
Dosage recommendations in the lower face
are given in Table 5.4.
As with onabotulinumtoxinA treatment,
the physician must have a thorough under-
standing of facial musculature and the bal-
ance between elevator and depressor mus-
cles. It is especially important in the lower
face to be familiar with the anatomy and
function of the facial muscles. Injections in
or near many perioral muscles can cause
facial asymmetry with expression. Because
the field of effect is influenced by volume,
the 1.5 cm3 dilution is recommended to limit
abobotulinumtoxinA effect to the muscles
injected.
AbobotulinumtoxinA can be used for ele-
vation of the oral commissure by injecting the
depressor angularis oris (DAO) with 10 DUs
or units of abobotulinumtoxinA. The men-
talis can be treated with 10 U to suppress chin
dimpling. Care is taken to keep both injec-
tions away from the depressor labialis inferi-
oris. Platysma injections for bands use 5 DUs
or units of abobotulinumtoxinA per injection
with a total of 20 U per band. This is a super-
ficial injection spaced out at 1-cm intervals
along the platysmal band.
As aesthetic physicians have become famil-
iar with this second botulinum toxin and
now a third botulinum toxin product in the
US, new innovations and techniques have
evolved, and new and interesting data have
emerged. It is with the large usage numbers
that we gain a further understanding of both
efficacy and safety.
References
Schantz EJ, Johnson EA. Botulinum toxin:
the story of its development for the
treatment of human disease. Perspect Biol
Med 1997 Spring;40(3):317–327.
Carruthers JA, Lowe NJ, Menter MA, et al.
BOTOX Glabellar Lines I Study Group. A
multicenter, double-blind, randomized,
placebo-controlled study of the efficacy and
safety of botulinum toxin type A in the
treatment of glabellar lines. J Am Acad
Dermatol 2002 Jun;46(6):840–849.
Botox Cosmetic [package insert]. Irvine,
Ca: Allergan, Inc; 2008.
Dysport [package insert]. Scottsdale, AZ:
Medicis Aesthetics Inc; 2009.
Simpson LL. Peripheral Actions of the
Botulinum Toxins. In LL Simpson (ed.)
Botulinum Neurotoxin and Tetanus Toxin.
San Diego, CA: Academic Press, 1989:
153–178.
Pickett AM, Hambleton P. Dose
standardization of botulinum toxin. Lancet
1994; 344:474–475.
Karsai S, Raulin C. Current evidence on the
unit equivalence of different botulinum
neurotoxin A formulations and
recommendations for clinical practice in
Dermatology. Dermatol Surg 2008;34:1–8.
Ascher B, Talarico S, Cassuto D, et al.
International consensus recommendations


Botulinum Toxins
on the aesthetic usage of botulinum toxin
type A (Speywood Unit)—Part II: Wrinkles
on the middle and lower face, neck and
chest. J Eur Acad Dermatol Venereol 2010
Nov;24(11):1285–1295.
Eisele KH, Taylor HV. Dissociation of the
900 kDa neurotoxin complex from c.
botulinum under physiological conditions.
Poster presented at: Toxins 2008; June
12–14, 2008; Baveno, Lake Maggiore, Italy.
Pickett A. Diffusion of Type A Botulinum
Toxin. Presented at the International
Masters Course on Aging Skin; January
8–11, 2009. Paris, France.
Hasegawa K, Watanabe T, Suzuki T, et al.
A novel subunit structure of clostridium
botulinum serotype D toxin complex with
three extended arms. J Biol Chem
2007;282(34):24777–24783.
Eisele KH, Fink K, Vey M, Taylor HV.
Studies on the dissociation of botulinum
neurotoxin type A complexes. Toxicon
2011 Mar 15;57(4):555–565.
Ascher B, Zakine B, Kestemont P, et al. A
multicenter, randomized, double-blind,
placebo-controlled study of efficacy and
safety of 3 doses of botulinum toxin type A
in the treatment of glabellar lines. J Am
Acad Dermatol 2004;51(2):223–233.
Vandenbergh PYK, Lison DF. Dose
standardization of botulinum toxin. Adv
Neurol 1998;78:231–235.
Kessler KR, Skutta M, Benecke R.
Long-term treatment of cervical dystonia
with botulinum toxin A: efficacy, safety, and
antibody frequency. German Dystonia
Study Group. J Neurol 1999
Apr;246(4):265–274.
Carruthers J, Lowe NJ, Mentor MA, Gibson
J. Double-blind placebo-controlled study of
the safety and efficacy of Botulinum toxin
type A for patients with glabellar lines.
Plastic Reconstr Surg 2003;112:1089–1098.
Monheit G, Carruthers A, Brandt F, Rand
R. A Randomized, double blind, placebo
controlled study of botulinum toxin A for
the treatment of glabellar lines:
Determination of optimal dose. Dermatol
Surg 2007 January;33:51–59.
Carruthers J, Fagien S, Matarasso SL.
Consensus recommendations on the use of
botulinum toxin type A in facial aesthetics.
Plast Reconstr Surg 2004;114 (Suppl 1):
S1–S22.
Ascher B, Rzany BJ, Grover R. Efficacy and
safety of botulinum toxin type A in the
treatment of lateral crow’s feet: double-
blind, placebo-controlled, dose-ranging
study. Dermatol Surg 2009;35:1478–1486.
Monheit G, Cohen J, Reloxin
Investigational Group. Long-term safety of
repeated administration of a new
formulation of botulinum toxin type A in
the treatment of glabellar lines: Interim
analysis from an open-label extension
study. J Am Acad Derm 2009 Sept;61(3):
421–425.
Rubin M, Dover J, Glogau R, et al. The
efficacy and safety of a new US botulinum
toxin type A in the retreatment of glabellar
lines following open-label treatment.
J Drugs Dermatol 2009;8(5):439–444.
Kane MA, Brandt F, Rohrich RJ, et al.;
Reloxin Investigational Group. Evaluation
of variable-dose treatment with a new U.S.
Botulinum Toxin Type A (Dysport) for
correction of moderate to severe glabellar
lines: results from a phase III, randomized,
double-blind, placebo-controlled study.
Plast Reconstr Surg 2009 Nov;124(5):
1619–1629. doi: 10.1097/PRS
.0b013e3181b5641b.
Hambleton P, Pickett AM. Potency
equivalence of botulinum toxin
preparations. J R Soc Med 1994
Nov;87(11):719.
Karsai S, Raulin C. Current evidence on the
unit equivalence of different botulinum
neurotoxin A formulations and
recommendations for clinical practice in
dermatology. Dermatol Surg 2009;35(1):
1–8.
Sampaio C, Costa J, Ferreira JJ. Clinical
comparability of marketed formulations of
botulinum toxin. Mov Disord 2004 Mar;19
Suppl 8:S129–S136.
Karsai S, Adrian R, Hammes S, et al. A
randomized double-blind study of the

5 Abobotulinumtoxin: Development and Aesthetic Usage

effect of Botox and Dysport/Reloxin on
forehead wrinkles and electromyographic
activity. Arch Dermatol 2007
Nov;143(11):1447–1449.
Lowe P, Patnaik R, Lowe N. Comparison of
two formulations of botulinum toxin type
A for the treatment of glabellar lines: A
double-blind, randomized study. J Am
Acad Dermatol 2006;55(6):975–980.
Cliff SH, Judodihardjo H, Eltringham E.
Different formulations of botulinum toxin
type A have different migration
characteristics: a double-blind, randomized
study. J Cosmet Dermatol. 2008;7(1):50–54.
Trindade De Almeida AR, Marques E, et al.
Pilot study comparing the diffusion of two
formulations of botulinum toxin type A in
patients with forehead hyperhidrosis.
Dermatol Surg 2007;33:S37–S43.
Hexsel D, Dal’Forno T, Hexsel C, et al. A
randomized pilot study comparing the
action halos of two commercial
preparations of botulinum toxin type A.
Dermatol Surg 2008;34(1):52–59.
Sugawara Y, Fujinaga Y. The botulinum
toxin complex meets E-cadherin on the
way to its destination. Cell Adh Migr 2011
Jan 8;5(1).
Sugawara Y, Matsumura T, Takegahara Y,
et al. Botulinum hemagglutinin disrupts
the intercellular epithelial barrier by
directly binding E-cadherin. J Cell Biol
2010 May 17;189(4):691–700.
Ascher B, Zakine B, Kestemont P, et al. A
multicenter, randomized, double-blind,
placebo-controlled study of efficacy and
safety of 3 doses of botulinum toxin type A
in the treatment of glabellar lines. J Am
Acad Dermatol 2004;51(2):223–233.
Kane MA, Brandt F, Rohrich RJ, et al.
Evaluation of variable-dose treatment with
a new U.S. Botulinum Toxin Type A
(Dysport) for correction of moderate to
severe glabellar lines: results from a phase
III, randomized, double-blind,
placebo-controlled study. Plast Reconstr
Surg 2009 Nov;124(5):1619–1629.
Dysport [package insert]. Scottsdale,
Arizona: Medicis Aesthetics Inc.; 2009.
Wortzman MS, Pickett A. The science and
manufacturing behind botulinum
neurotoxin type A-ABO in clinical use.
Aesth Surg J 2009;29(6, Suppl 1):
S34–S42.
Pickett A, Perrow K. Formulation
composition of botulinum toxins in clinical
use. J Drugs Dermatol 2010
Sep;9(9):1085–1091.
Botox cosmetic [package insert]. Irvine,
CA: Allergan, Inc; 2002.
Xeomin cosmetic [package insert].
Frankfurt, Germany. Merz
Pharmaceuticals; 2010.
Carruthers A, Carruthers J. Botulinum
toxin products overview. Skin Ther Lett
2008 Jul–Aug;13(6):1–4.



IncobotulinumtoxinA (Xeomin®/Bocouture®)
Ulrich K¨uhne, MD (DALM) and Matthias Imhof, MD (DALM)
Board Certified Dermatologist and Allergologist, Aesthetische Dermatologie im Medico Palais, Bad Soden, Germany
Introduction
IncobotulinumtoxinA (Merz Pharmaceuti-
cals GmbH, Germany) is a botulinum neuro-
toxin type A (BoNTA) preparation, which
represents a new development in the rapidly
expanding field of botulinum toxin ther-
apies. In contrast to other commercially
available preparations, incobotulinumtoxinA
is free from complexing proteins. It has
shown good efficacy and tolerability in a
range of therapeutic and aesthetic appli-
cations. It is approved for the treatment
of blepharospasm, post-stroke upper-limb
spasticity, and cervical dystonia in several
countries under the trade name Xeomin®
or Xeomeen®. It also received approval
in August 2010 from the Food and Drugs
Administration
(FDA)
for
treatment
of
blepharospasm and cervical dystonia in the
United States of America. In the aesthetic
setting, incobotulinumtoxinA is currently
licensed for use in the treatment of glabellar
frown lines, lateral periorbital lines (crow’s
feet) and upper facial lines in Europe under
the trade name Bocouture®. The indication
glabellar frown lines alone is approved in the
USA and Canada as well as in several other
countries in Latin America and Asia.
This chapter includes a discussion of
the pharmacological properties of incobo-
tulinumtoxinA and how being free of com-
plexing proteins could hold clinical relevance.
The use of incobotulinumtoxinA in aesthetic
applications is also discussed.
Manufacture and production of
incobotulinumtoxinA
Free from complexing proteins
IncobotulinumtoxinA
is
produced
from
Clostridium botulinum type A, Hall strain,
which is fermented under anaerobic con-
ditions. This process yields the 150 kDa
neurotoxin heavy and light chains, linked
by a disulfide bridge, in the context of a
large molecular weight complex containing
other clostridial proteins. Thereafter, several
purification steps are performed to remove
nucleic acids, hemagglutinins, and other
contaminants, to yield only the 150 kDa
active neurotoxin free from complexing
proteins (Figure 6.1). OnabotulinumtoxinA
(Allergan, Irvine, CA, USA) is produced
by repeated cycles of precipitation and
redissolution,
while
abobotulinumtoxinA
(Ipsen Ltd, Berkshire, UK) is produced by a
column separation method of purification
[1]. Both processes yield products with toxin
complexes of different sizes [2].
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:


Botulinum Toxins
Figure .Most
commercially available
BoNTA preparations consist
of the neurotoxin and
associated complexing
proteins (a). The manufacture
of incobotulinumtoxinA
removes the complexing
proteins (b) to leave only the
pure active neurotoxin (c)
(reproduced with permission
of Merz Pharmaceuticals
GmbH).
Free from denatured or inactive neurotoxin
In addition to being free from complex-
ing proteins, incobotulinumtoxinA contains
the smallest amount of neurotoxin protein.
Using rabbit and guinea pig antibodies raised
against the 150 kDa neurotoxin purified from
C. botulinum type A strain ATCC 3502 (Hall
strain), a sensitive sandwich ELISA was per-
formed to determine the mean concentration
of the 150 kDa neurotoxin present in differ-
ent batches of onabotulinumtoxinA, abobo-
tulinumtoxinA and incobotulinumtoxinA [3].
The mean concentration was then used to
calculate the biological activity (units, U) per
ng of neurotoxin protein, which is a mea-
sure of the specific neurotoxin potency. The
mean concentration of 150 kDa neurotoxin
per 100 U was 0.73 ng for onabotulinumtox-
inA, 0.65 ng for abobotulinumtoxinA, and
0.44 ng for incobotulinumtoxinA. The result-
ing specific potency was calculated to be
137, 154, and 227 g/ng for onabotulinum-
toxinA, abobotulinumtoxinA, and incobo-
tulinumtoxinA, respectively [3], showing that
incobotulinumtoxinA had the highest spe-
cific neurotoxin activity. However, it should
be noted that the units of abobotulinumtox-
inA cannot be directly compared to the units
of onabotulinumtoxinA and incobotulinum-
toxinA and therefore an assessment of the
amount of inactive neurotoxin in abobo-
tulinumtoxinA, compared to onabotulinum-
toxinA or incobotulinumtoxinA, cannot be
made. The implication of these results, given
that clinical trials show similar efficacy of
onabotulinumtoxinA and incobotulinumtox-
inA when used at a 1 : 1 dose conversion ratio
[4–6], is that the 0.44 ng neurotoxin per 100 U
incobotulinumtoxinA has the same potency
as the 0.73 ng neurotoxin per 100 U onabo-
tulinumtoxinA, which suggests that onabo-
tulinumtoxinA may contain a considerable
amount of inactive or denatured neurotoxin.
Any inactive neurotoxin present, while inca-
pable of contributing to the therapeutic or
aesthetic effect, could contribute to the stim-
ulation of antibody production [3], which is
discussed in detail later. It has been reported
that the manufacturing process of incobo-
tulinumtoxinA does not lead to denaturation
or inactivation of the active toxin [3].
Implications of Being Free
from Complexing Proteins and
Inactive Neurotoxin in
IncobotulinumtoxinA
In the past, it was suggested that the complex-
ing proteins present in some commercially
available formulations of BoNTA played a
beneficial role in stabilizing the 150 kDa
active neurotoxin and limiting its migration.
While evidence suggests that the neurotoxin
must remain within the 900 kDa complex to
maintain its oral activity [7], when BoNTA
preparations are used for therapeutic or cos-
metic applications, complexing proteins are

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

pH 6.6
pH 6.75
pH 6.9
pH 7.2
0
10
20
30
40
50
60
70
80
90
100
Percent of neurotoxin release
Release after 1 min
Release after 15 min
Figure .Dissociation of the neurotoxin
from the complexing proteins occurs in
less than a minute at physiological pH
(reproduced with permission of Merz
Pharmaceuticals GmbH).
not only unnecessary for the desired effects,
but may serve to stimulate an unwanted
immune response.
Dissociation
The dissociation of the 150 kDa neurotoxin
from complexing proteins has been studied
by exposing the 900 kDa complex to vari-
ous pH values prior to elution of the dissoci-
ated complex from a chromatographic matrix
[8]. The eluted fractions were then analyzed
by Western blotting and for toxin activity.
At physiological pH, the 150 kDa neurotoxin
dissociated from the 900 kDa complex in less
than a minute (Figure 6.2). Considering that
the dissociation is so rapid, the complexing
proteins cannot stabilize the neurotoxin, nor
limit its spread. These results call into ques-
tion the necessity of the complexing proteins
in BoNTA formulations used for therapeutic
and cosmetic purposes.
Stability
The results described earlier suggest that
complexing proteins play little or no role in
stabilizing the neurotoxin after injection. Fur-
ther experiments have shown that complex-
ing proteins are also not required to stabilize
the lyophilized neurotoxin during long-term
storage at room temperature or short-term
storage at temperatures above 40◦C, prior
to reconstitution [9–11]. Incobotulinum-
toxinA was stored at temperatures up to 40◦C
with no significant reduction in the quality
of the product, as tested using standardized
analytical methods for the batch-release test-
ing of incobotulinumtoxinA. The results of
these experiments have shown that incobo-
tulinumtoxinA can be stored unopened at
room temperature for 3 years [10,12,13]. Fur-
thermore, no negative effects on the quality of
incobotulinumtoxinA were detected follow-
ing a temperature stress test, during which
vials of incobotulinumtoxinA were stored
at temperatures between 40◦C and 60◦C
for up to 1 month [10]. The efficacy of freshly
constituted and room-temperature-stored
incobotulinumtoxinA has been compared in
a split-face study in which 10 U was injected
into the crow’s feet of 21 subjects [11].
Over 4 months of follow-up, there was no
statistically significant difference in either
efficacy or longevity between the fresh and
stored products. So not only are complexing
proteins unnecessary to maintain the stabil-
ity of the neurotoxin at room temperature
and higher, but incobotulinumtoxinA can
be stored without refrigeration, unlike other
BoNTA preparations [14–16], a property
that is of practical benefit to physicians.
Onset and duration of effect
The possibility that some BoNTA prepara-
tions contain denatured or inactive neuro-
toxin in addition to active neurotoxin may
result in a slower onset of effect as these com-
ponents may compete with the active neuro-
toxin for binding sites on the surface of nerve
terminals at the neuromuscular junction. The


Botulinum Toxins
onset and duration of effect of BoNTA prepa-
rations have been compared in a study of 180
subjects treated for glabellar frown lines who
were randomized to treatment with incobo-
tulinumtoxinA (21 U), onabotulinumtoxinA
(21 U) or abobotulinumtoxinA (63 U) [17].
For both men and women, a significantly
earlier time to onset of treatment effect as
well as a longer duration of effect was seen
for incobotulinumtoxinA compared with the
other BoNTA preparations.
Neurotoxin spread
Migration of neurotoxin into untargeted
muscles can cause unwanted effects and
adverse events, especially in cosmetic appli-
cations of botulinum toxins where muscles
are in close proximity [18]. The dissocia-
tion of active neurotoxin from the complex-
ing proteins is very rapid at physiological pH,
so the complexing proteins are unlikely to
have an impact on the spread of the toxin [8],
although this remains a controversial topic
[19]. This is supported by studies demonstrat-
ing that the mean anhidrotic areas induced by
preparations with complexing proteins com-
pared with those without are not statistically
significantly different sizes, indicating that
the migratory potential of the neurotoxin is
independent of the presence or absence of
complexing proteins [20].
This is supported by data from a histo-
logical assessment of diffusion, which exam-
ined neural cell adhesion molecule (N-CAM)
expression following injection of onabo-
tulinumtoxinA,
abobotulinumtoxinA,
or
incobotulinumtoxinA into the mouse leg
[21]. N-CAM is expressed after denervation
and therefore its appearance is an indication
of BoNTA activity. All three products dis-
played modest diffusion into nearby muscles
and abobotulinumtoxinA showed a wider
spread than onabotulinumtoxinA or incobo-
tulinumtoxinA [21]; however, the ratio of
onabotulinumtoxinA :
incobotulinumtox-
inA : abobotulinumtoxinA used was 1 : 1 : 4.
There is some evidence to suggest that this
was too high for abobotulinumtoxinA and
could have accounted for the wider spread of
abobotulinumtoxinA in this study. Varying
conversion ratios could also explain the
contradictory results concerning the relative
spread of BoNTA preparations with com-
plexing proteins seen in various other studies
[22–26]. One study recommended using an
onabotulinumtoxinA : abobotulinumtoxinA
conversion ratio of not more than 1 : 2 [24],
although a ratio of 1 : 2.5 is commonly used
in clinical practice.
Several studies indicate that the absence of
complexing proteins in incobotulinumtoxinA
has no influence on the spread of neurotoxin
to adjacent muscles. A double-blind, ran-
domized, single-center trial in 32 healthy
male volunteers measured the contraction
of muscles adjacent to those targeted by
injection
after
treatment
with
incobo-
tulinumtoxinA and onabotulinumtoxinA in
an intra-individual comparison design where
equivalent doses of the toxins (either 2, 4, 16
or 32 Units) were injected into the extensor
digitorum brevis (EDB) muscles of contralat-
eral feet [27]. Local diffusion was assessed
by measuring the compound muscle action
potential (CMAP) M-wave amplitude in two
muscles adjacent to the EDB – the abductor
digiti quinti (ADQ) and the abductor hallucis
(AH) muscle – 4 weeks postinjection relative
to baseline. The results showed that, for all
dose groups, the CMAP M-wave amplitude
was more than 80% the baseline value in both
ADQ and AH muscles at week 4 and all sub-
sequent postinjection visits, despite a marked
paretic effect on the injected EDB muscle.
There were no statistically significant dif-
ferences between the mean CMAP M-wave
amplitudes obtained after incobotulinum-
toxinA and onabotulinumtoxinA treatment
in any dose group [27].
A comparison of incobotulinumtoxinA and
abobotulinumtoxinA in the forehead of 80
subjects using a conversion ratio of 1.0 : 2.5 U
found a larger field of anhidrotic effects with
abobotulinumtoxinA, but similar results for
fields of muscular effects [28].
The influence of injection volume on
neurotoxin diffusion was investigated in a

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

prospective, randomized, controlled study in
10 volunteers with dynamic forehead lines
who were given two injections, one on each
side of their forehead 2.5 cm above the bony
orbital rim [29]. The same dose of the same
BoNTA (2 U) was used, but administered
in different injection volumes (0.25 mL and
0.05 mL). The total area affected was assessed
by visual inspection of the subject’s fore-
head during active muscle contraction. In this
study, it was shown that the greater injec-
tion volume affected an area 50% larger than
that of the smaller injection volume in nine
of the ten subjects [29]. This suggests that
injection volume can have an impact on
BoNTA migration.
While contradictory results have been
published about the relative spread of dif-
ferent botulinum toxin formulations, a com-
parison of incobotulinumtoxinA with onabo-
tulinumtoxinA that used identical injection
doses and volumes showed no difference in
anhidrotic halo area in 20 subjects at 6 weeks,
whereas abobotulinumtoxinA, using a con-
version ratio of 1 : 2.5, produced a statistically
significantly greater maximal area of anhidro-
sis within 6 weeks [20]. This result suggests
that factors such as injection volume and dose
have a larger impact on spread than the pres-
ence or absence of complexing proteins. In
cosmetic applications of BoNTAs, especially
in the face, precise localization of the neuro-
toxin, and hence its paretic effect, is essen-
tial to obtain the desired aesthetic enhance-
ment while avoiding unwanted effects due to
the action of the neurotoxin on untargeted
muscles.
Immunogenicity
A further implication of freedom from com-
plexing proteins in the formulation of incobo-
tulinumtoxinA is the reduced potential for
immunogenicity. The 900 kDa complex com-
pared to the 150 kDa neurotoxin represents
a substantial additional foreign protein load.
Complexing proteins may increase the risk of
producing neutralizing antibodies against the
active neurotoxin. For example, the effect on
antibody production induced by neurotoxin
alone or neurotoxin plus complexing proteins
was investigated in a mouse study using a
botulinum toxin type B preparation (BoNTB)
[30]. The amount of antibodies produced was
greater when complexing proteins (hemag-
glutinin subcomponents, HA1 and HA3b)
were present compared with when the neu-
rotoxin alone was administered. Moreover,
the original formulation of onabotulinumtox-
inA was six times more likely to induce anti-
body production than the more recent for-
mulation (p < 0.004), which contains 80% less
clostridial protein than its predecessor [31].
This led the investigators to conclude that the
reduced immunogenicity observed was asso-
ciated with reduced protein load [31].
The development of neutralizing antibod-
ies is particularly important, given that their
presence can lead to treatment failure and
termination. Risk factors for antibody for-
mation include a shorter injection inter-
val, a larger number of booster injections,
and higher doses per injection [32]. These
are important considerations as patients
look to receive long-term treatment, often
involving many injections. While the inci-
dence of antibody-induced treatment failure
is reported to be low for cosmetic appli-
cations, cases have been reported in der-
matological applications [33, 34]. Since the
original formulation of onabotulinumtoxinA
was changed to reduce the protein load per
dose, the incidence of neutralizing antibody
development has been significantly reduced
[31, 35], but cases still occur. A 48-year-old
woman who had been treated successfully
for glabellar frown lines and lateral perior-
bital wrinkles with 14 injections of onabo-
tulinumtoxinA with a duration of response of
between 3 and 4 months, failed to respond
to the 15th injection [36]. Subsequent injec-
tions of BoNTA yielded no effect but a
BoNTB preparation was effective. Analysis
showed the presence of neutralizing antibod-
ies to BoNTA in the patient’s blood [36].
Another report describes the case of a 20-
year-old woman who developed antibody-
induced treatment failure after the fourth


Botulinum Toxins
injection series using onabotulinumtoxinA
for the treatment of masseteric hypertro-
phy [37]. Sixty units were injected at each
series and there was a 4- to 5-month inter-
val between injections. A separate paper
describes five case studies in which neu-
tralizing antibodies to both onabotulinum-
toxinA and abobotulinumtoxinA developed
after injection for aesthetic use and resulted
in secondary treatment failure [34]. In one
of the case studies, the subject was given
a final treatment of incobotulinumtoxinA
after previous failure on abobotulinumtox-
inA and onabotulinumtoxinA, but the dura-
tion of effects was only 2 weeks. This result
was expected given that the subject already
had existing neutralizing antibodies and sec-
ondary treatment failure. Two similar cases
have been reported with incobotulinum-
toxinA for therapeutic use in patients with
dystonia pretreated with abobotulinumtox-
inA [38, 39]. To date, no cases of secondary
treatment failure with incobotulinumtoxinA
have been reported in toxin-na¨ıve patients.
There is no doubt that the lower doses
of BoNTAs generally used in cosmetic com-
pared to therapeutic applications pose a
lower risk towards the generation of neu-
tralizing antibodies; however, emerging case
reports suggest that it is not an issue that can
be dismissed. This is especially true as the
reduced protein load in the newer formula-
tion of onabotulinumtoxinA does not elim-
inate the potential of inducing an immune
response [37, 40]. Thus, the freedom from
complexing proteins of incobotulinumtoxinA
may present a clinical advantage in terms
of reduced potential for eliciting an immune
response.
IncobotulinumtoxinA is not associated
with antigenicity in animal models [41]. The
development of neutralizing antibodies in
New Zealand white rabbits (n = 60) was
assessed
following
repeated
intradermal
administration
of
incobotulinumtoxinA,
abobotulinumtoxinA, and onabotulinumtox-
inA [41]. Each animal received eight admin-
istrations of approximately 5.34 LDU/kg at
2- to 8-week intervals [41]. Results demon-
strated that incobotulinumtoxinA, unlike
abobotulinumtoxinA and onabotulinumtox-
inA, was not associated with the formation
of neutralizing antibodies [41]. This finding
is particularly pertinent, considering that the
doses used were comparatively higher than
those that would be used in human subjects
[42]. Long-term study data in humans to look
at efficacy and safety of incobotulinumtoxinA
are becoming available. In a study of patients
with upper-limb spasticity over the course of
up to 89 weeks, patients received an injection
of either incobotulinumtoxinA or placebo
followed by up to five injections of incobo-
tulinumtoxinA in an open-label extension
phase. No patient developed neutralizing
antibodies to incobotulinumtoxinA (assessed
using the hemidiaphragm assay) throughout
the study [43], which supports the results in
animal models.
In summary, the development of neutral-
izing antibodies remains an issue in aesthet-
ics and should be considered when select-
ing treatment. Several reports have shown
that neutralizing antibodies are raised in
patients receiving BoNTA formulations con-
taining complexing proteins, despite the
reduction of protein load in the newer for-
mulation of onabotulinumtoxinA [36,37,40].
As such, the future of cosmetic dermatol-
ogy may shift towards using products which
are free from complexing proteins entirely,
and with the least potential for inducing an
immune response. It is worth remembering
that antibody-induced secondary treatment
failure could have important repercussions
for a patient should they find themselves in
need of therapeutic BoNTA treatment in later
life, for example, for post-stroke spasticity.
Evidence for Equipotency
between IncobotulinumtoxinA
and OnabotulinumtoxinA
To ensure safe and effective dosing, each
batch of BoNTA must be tested for potency
and stability before it can be released onto
the market and applied for human use.
The potencies of incobotulinumtoxinA and

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

onabotulinumtoxinA have been tested in
product-specific mouse LD50 assays, leading
to discussion of whether differences in
potency may exist between the two products.
Using the batch-release mouse LD50 assay for
incobotulinumtoxinA, five unopened batches
of incobotulinumtoxinA and onabotulinum-
toxinA were compared. The results showed
that the potencies of incobotulinumtoxinA
(103.0
±
5.7)
and
onabotulinumtoxinA
(101.7 ± 6.2) were not statistically signifi-
cantly different (p = 0.734) [44]. The authors
concluded that the biological potencies of
incobotulinumtoxinA
and
onabotulinum-
toxinA are equivalent and that a 1 : 1 dose
conversion ratio is appropriate in clinical
practice [44]. As required by governmental
agencies, the LD50 assay is being replaced
by more humane, cell-based assays, which
must be cross-validated against the LD50
assay to provide the same potency result.
Each manufacturer is developing their own
proprietary cell-based assay [45,46].
The dose conversion ratio established in
the earlier biological potency study has been
borne out in clinical studies published to date.
A randomized, controlled phase I study com-
pared the efficacy and tolerability of equal
doses of incobotulinumtoxinA and onabo-
tulinumtoxinA injected into the EDB mus-
cle of 14 healthy volunteers [47]. Based on
the EDB test, no statistically significant dif-
ferences were found between incobotulinum-
toxinA and onabotulinumtoxinA for time to
onset of action, response rate, CMAP ratios,
and tolerability [47]. The between-treatment
comparison showed that incobotulinumtox-
inA was at least as effective as onabotulinum-
toxinA in paralyzing EDB muscle activity
over the study period.
In
comparative
phase
III
studies
in
therapeutic indications using a 1 : 1 dose
conversion ratio of incobotulinumtoxinA :
onabotulinumtoxinA, incobotulinumtoxinA
demonstrated comparable efficacy and safety
to onabotulinumtoxinA, further supporting
the preclinical equipotency data. In a large,
randomized, actively controlled study in 463
patients with cervical dystonia, the aver-
age improvement on the Toronto Western
Spasmodic Toricollis Rating Scale at day
28 posttreatment compared with baseline
was
−6.6
for
incobotulinumtoxinA
and
−6.4 for onabotulinumtoxinA [4]. Similarly,
in a double-blind phase III trial in 304
patients with blepharospasm comparing the
effect of incobotulinumtoxinA with that of
onabotulinumtoxinA, there were no statisti-
cally significant differences found between
incobotulinumtoxinA
and
onabotulinum-
toxinA for any efficacy or safety variable
[5]. In aesthetics, the efficacy of incobo-
tulinumtoxinA has been compared to that of
onabotulinumtoxinA in several head-to-head
trials using a 1 : 1 dose conversion ratio and
showed comparable efficacy [6,48,49].
A French consensus, based on a biolog-
ical, bibliographic, and clinical analysis of
incobotulinumtoxinA and onabotulinumtox-
inA, concluded that the two toxins are clin-
ically equivalent for efficacy and safety, and
that a switch from one drug to the other
can be made using a simple 1 : 1 conversion
ratio [50]. A German retrospective analysis
of clinical practice also confirmed the results
of prospective clinical trials by demonstrat-
ing that, in daily practice, incobotulinumtox-
inA and onabotulinumtoxinA are used at a
1 : 1 dose ratio and display comparable effi-
cacy and safety [51]. A meta-analysis of clin-
ical studies that directly compared incobo-
tulinumtoxinA and onabotulinumtoxinA for
aesthetic indications also concluded that
there was no difference in relative potency
of the two products [52]. In summary, evi-
dence from clinical trials supports the pre-
clinical data that incobotulinumtoxinA and
onabotulinumtoxinA have equal biological
potency and a 1 : 1 dose conversion ratio is
appropriate.
Aesthetic Applications of
IncobotulinumtoxinA
Glabellar Frown Lines
IncobotulinumtoxinA was approved by The
Federal Institute for Drugs and Medical


Botulinum Toxins
Devices (Bundesinstitut f¨ur Arzneimittel und
Medizinprodukte [BfArM]) in 2009 for the
temporary improvement in the appearance
of moderate to severe glabellar lines seen at
frown in adults <65 years, when the severity
of these lines has an important psychologi-
cal impact for the patient. The recommended
injection volume is 0.1 ml (4 U) injected into
each of five injection sites: two in each corru-
gator muscle and one in procerus muscle for a
total dose of 20 U. FDA approval was received
in July 2011 based on the results of two piv-
otal placebo-controlled trials that used the
new more stringent FDA responder defini-
tion of an improvement of at least 2 points
from baseline as assessed by both investiga-
tor and subject on separate 4-point scales.
In both studies, the primary composite effi-
cacy endpoint at maximum frown on Day 30
was achieved in a significantly higher number
of patients treated with incobotulinumtox-
inA (47.8% and 60.3%) than placebo, none of
whom achieved the composite endpoint [53,
54]. To allow comparisons with earlier stud-
ies, a recent post hoc study has pooled the
data from these two trials and analyzed them
using the previous FDA responder defini-
tion: subject with at least a 1-point improve-
ment on the Facial Wrinkle Scale (FWS).
Responder rates were significantly greater
for incobotulinumtoxinA than placebo at all
follow-up visits (p < 0.0001) [55]. Incobo-
tulinumtoxinA achieved a maximum respon-
der rate of 93.1% at 30 days and a long dura-
tion of treatment effect: 45.7% of subjects
showed efficacy at 4 months.
Various other placebo-controlled, open-
label and head-to-head phase III studies have
evaluated the efficacy and safety of incobo-
tulinumtoxinA for the treatment of glabellar
frown lines [6,56–61].
In
a
head-to-head
study
that
used
unmatched doses of each product, the
authors concluded that 20 U onabotulinum-
toxinA was as effective as 30 U of incobo-
tulinumtoxinA in reducing the severity of
glabellar frown lines at 28 days postinjection,
despite the 50% difference in unit doses
administered [58]. Using a similar study
design, the reverse has also been shown to
be true [59]. Active comparator should be
given at the optimal dose. It had already been
established and reflected in the SPCs that
the correct starting dose for incobotulinum-
toxinA (and onabotulinumtoxinA) in the
treatment of moderate to severe glabellar
frown lines is 20 U [12, 13, 15]. There is little
or no incremental clinical benefit in start
doses >20 U for either product [62,63].
The efficacy of two treatments for the
same indication can only be directly com-
pared in head-to-head comparative random-
ized studies using optimal doses. A large,
European head-to-head study investigated
the non-inferiority of incobotulinumtoxinA
to onabotulinumtoxinA, when a 1 : 1 dose
ratio was used [6]. Female subjects with mod-
erate to severe glabellar frown lines (sever-
ity score of 2–3 on FWS, as assessed by
the investigator) were randomized to receive
24 U of incobotulinumtoxinA (n = 284) or
onabotulinumtoxinA (n = 97). At week 4
postinjection, response at maximum frown –
defined as an improvement of at least 1 point
on the FWS compared with baseline – was
96.4% and 95.7% in the incobotulinumtox-
inA and onabotulinumtoxinA groups, respec-
tively, as assessed by an independent asses-
sor from standardized clinical photographs
(Figure 6.3a and Figure 6.4a,b). Therapeutic
effect was maintained at week 12, with 80.1%
of subjects in the incobotulinumtoxinA group
and 78.5% of subjects in the onabotulinum-
toxinA group defined as responders at maxi-
mum frown (Figure 6.3a). Similar results were
observed by the investigator (Figure 6.3b).
Importantly, the subjects themselves rated
their treatment highly on the patient’s global
assessment and the incidence of adverse
events throughout the study was low. This
study confirmed that incobotulinumtoxinA is
non-inferior to onabotulinumtoxinA for the
treatment of glabellar frown lines and is well
tolerated [6].
To extend and confirm the results of
this non-inferiority trial, a new US head-
to-head study, this time powered to test
equivalence, randomized 250 subjects in a

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

100
(a)
Responders (%)
0
Week 4
Week 12
20
40
60
80
OnabotulinumtoxinA
IncobotulinumtoxinA
OnabotulinumtoxinA
IncobotulinumtoxinA
100
(b)
Responders (%)
0
Week 4
Week 12
20
40
60
80
Figure .Percentage of responders at maximum frown at weeks 4 and 12 according to the facial wrinkle scale
for the per protocol set. (a) Independent rater assessment based on digital photographs. (b) Investigator
assessment based on the live patient. Source: Sattler 2010 [6]. Reproduced with permission of Wolters Kluwer
Health, Inc.
1 : 1 ratio to incobotulinumtoxinA or onabo-
tulinumtoxinA, with both treatments given at
their optimal dose of 20 U for the treatment
of moderate-to-severe glabellar frown lines
[49]. Primary efficacy analysis showed a very
high and statistically equivalent response rate
for incobotulinumtoxinA and onabotulinum-
toxinA at 1 month (95.7% and 99.2%, respec-
tively). Similar efficacy profiles were demon-
strated at 1, 2, 3, and 4 months whether
assessed by independent raters or treating
physicians. In the US head-to-head study,


Botulinum Toxins
Figure .Clinical photographs taken at maximum frown in patients treated with (a) incobotulinumtoxinA
and (b) onabotulinumtoxinA. Source: Sattler 2010 [6]. Reproduced with permission of Wolters Kluwer Health, Inc.
subject assessments of treatment satisfaction
with incobotulinumtoxinA were over 90%
throughout the study and similar to onabo-
tulinumtoxinA at all timepoints, thus con-
firming results from the European study.
In an open-label study of 105 patients with
moderate or severe glabellar frown lines,
patients received 20 U incobotulinumtoxinA
(0.1 ml to each of five injection sites) and were
assessed at week 4 and week 12 postinjection.
Response at maximum frown was 98.1% at
week 4 and 80.0% at week 12. Adverse events
were low and no patients developed neutral-
izing antibodies. The authors concluded that
incobotulinumtoxinA is effective and well
tolerated in the treatment of glabellar frown
lines [57].
Men typically have a stronger glabellar
muscle
mass
than
women
and
require
higher doses. In a small study of 12 men,
the effectiveness of BoNTA preparations for
hyperdynamic forehead lines was similar
when administered at a dose ratio of 1 : 1 : 3
onabotulinumtoxinA :
incobotulinumtox-
inA : abobotulinumtoxinA [64]. A study of
29 subjects with Fitzpatrick skin types IV to
VI has confirmed that the efficacy and safety
of incobotulinumtoxinA for the correction
of glabellar lines is similar to that among
individuals with fairer skin [61].
A prospective, open-label, multicenter,
repeat-dose, phase III trial has investi-
gated the long-term safety and efficacy of
incobotulinumtoxinA for the treatment of
glabellar frown lines over 2 years and up to 8
injection cycles in 796 subjects [60]. Efficacy
was assessed by percentage of responders
at maximum frown (score of 0 [none] or
1 [mild]) on the FWS at day 30 of each
treatment cycle. Responder rates remained

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

stable during cycles 1–7 at approximately
80% (range 79.1−82.7%), and were higher
in cycle 8 (89.6%). Results from the subject
assessment on a 4-point scale at maximum
frown (subjects with ≥1-point improvement)
also showed a maintained response to repeat
doses over time [60].
Crow’s Feet
Two head-to-head studies have compared the
efficacy of incobotulinumtoxinA and onabo-
tulinumtoxinA in the treatment of crow’s
feet [48, 65]. In the first study, 21 patients
with a score of 2 or 3 on the FWS (moder-
ate or severe) received treatment for crow’s
feet with 12 U of either onabotulinumtox-
inA or incobotulinumtoxinA in an intra-
individual split-face, double-blind, proof-of-
concept study. At 1 month postinjection,
the response rates were 95% for the side
of the face treated with incobotulinumtox-
inA and 90% for the onabotulinumtoxinA-
treated side. The study showed that there
were no statistically significant differences in
response rates between the two products over
the entire observation period of 4 months,
supporting the theory that the two products
are equipotent and that the commonly used
1 : 1 dose conversion ratio is appropriate. Tol-
erability was also rated as ‘very good’ by most
patients over the entire study period, leading
the authors to conclude that both products
are well-tolerated and effective treatments for
periorbital wrinkles [48].
The
second
study
used
a
split-face,
crossover design to compare incobotulinum-
toxinA and onabotulinumtoxinA at a 1 : 1
dose conversion ratio in two consecutive
treatment cycles, each of 3 months, sepa-
rated by 6 months [65]. The study recruited
14 women with symmetrical crow’s feet
with a score of 1 to 4 on the Merz 5-point
scale at maximum contraction. Using the
same injection sites and techniques, 12 U
of incobotulinumtoxinA or onabotulinum-
toxinA were injected into the lateral peri-
orbital region of each eye. Both treatments
were similarly effective in reducing the sever-
ity of crow’s feet at rest and at maximum
contraction, in both treatment cycles, and on
both sides of the face.
A randomized, split-face study has also
compared the safety and efficacy of incobo-
tulinumtoxinA
and
abobotulinumtoxinA
for the treatment of crow’s feet at a dose
conversion ratio of 1 : 3 [66]. Twenty women
with moderate-to-severe crow’s feet at rest
according to the Merz Aesthetics scale
received 9 U incobotulinumtoxinA or 27 U
abobotulinumtoxinA to the lateral perior-
bital region. Both products demonstrated
comparable efficacy with responder rates
(≥1-point improvement from baseline) of
100% and 83% at 2 weeks and 4 months
posttreatment, respectively. At 6 months
posttreatment, responder rates were 67%
and
61%
for
incobotulinumtoxinA
and
abobotulinumtoxinA, respectively. In this
study the clinical data were supported by
electromyography findings, which showed
that both treatments induced similar levels
of neuromuscular blockade. In addition,
the
electromyographic
values
recorded
6 months posttreatment, although higher
when compared with the 4-month data, were
still lower than baseline values, indicating a
partial persistence of the therapeutic effect
at 6 months posttreatment.
Upper Facial Lines
The efficacy and safety of incobotulinum-
toxinA for upper facial lines, a combination
of glabellar frown lines (GFL), horizontal
forehead lines (HFL), and lateral perior-
bital lines (LPL) has been assessed in two
studies [67, 68]. In a placebo-controlled,
double-blind
study,
156
subjects
with
moderate-to-severe upper facial lines on the
Merz Aesthetics scales were randomized to
incobotulinumtoxinA (n = 105) or placebo
(n = 51). IncobotulinumtoxinA was admin-
istered as doses of 54–64 U as follows: GFL
20 U, HFL 10–20 U, and LPL 24 U. Com-
pared with placebo, incobotulinumtoxinA
demonstrated significantly greater efficacy


Botulinum Toxins
in treating upper facial lines separately and
combined. Investigator-assessed scores of 0
(no lines) and 1 (mild) on day 30 at maximum
contraction for incobotulinumtoxinA versus
placebo were: GFL (84.5% versus 0.0%), HFL
(70.9% versus 2.1%), LPL (64.1% versus 2.1%)
and upper facial lines combination (55.3%
versus 0.0%), all (p ≤0.0001).
In the second study, 27 women were treated
for upper facial lines as follows: GFL 10−30 U,
LPL 10−30 U, and HFL 6−15 U [68]. Wrin-
kle severity was assessed at days 0 (base-
line), 2, 7, 14, 28, and 112 using the Merz
Aesthetics scales. Responders were defined
as those with an improvement from base-
line of at least 1 point. IncobotulinumtoxinA
had a rapid onset of action with a notice-
able decrease in the mean wrinkle severity
score from day 2. Responder rates for the dif-
ferent indications generally peaked at days
7 to 14. At 112 days, mean wrinkle sever-
ity scores remained significantly lower than
baseline (p < 0.05). Patients’ self-perception
was greatly improved after treatment, with
beneficial effects lasting up to 4 months after
treatment.
Platysmal Bands
Two studies have evaluated the efficacy of
incobotulinumtoxinA in the treatment of
platysmal bands. In the first study, 25 sub-
jects with platysmal bands scoring 2 or 3 on
the validated 5-point Merz dynamic platys-
mal bands scale [69] received a total of 60 U
incobotulinumtoxinA divided between four
platysmal bands (20 U in both medial bands
and 10 U in both lateral bands) [70]. In the
second study, 23 subjects with 2 to 4 platys-
mal bands scoring ≥1 on the Merz platys-
mal band scale received 15 U incobotulinum-
toxinA in each band [71]. In both studies, a
responder was defined as a subject with at
least a 1-point improvement from baseline on
the dynamic platysmal bands scale. Response
rates at maximum contraction of 100% were
achieved at day 14 in the first study and day 8
in the second study. Response rates remained
high at the end of follow-up and treatment
was well tolerated in both trials. Further stud-
ies investigating the use of incobotulinumtox-
inA in a range of aesthetic applications are
ongoing.
Personal Experience
In our practice for aesthetic dermatology,
we have been using incobotulinumtoxinA for
aesthetic indications since 2005, after having
mainly used onabotulinumtoxinA before. We
could not observe any difference in efficacy,
tolerability, and patient satisfaction between
the two products. Besides the standard indi-
cations such as glabellar lines, horizontal
frown lines, crow’s feet, and more advanced
indications such as brow lift, perioral lines,
cobblestone chin, lifting of oral commissures,
and softening platysmal bands, we also use
incobotulinumtoxinA in formerly forbidden
zones such as the lower lateral forehead,
lower eyelids, and over the zygoma. In these
areas, we use an intradermal injection tech-
nique with multiple injection points and very
small aliquots (0.3–0.5 U of incobotulinum-
toxinA) (Figure 6.5) [72].
Summary
In conclusion, incobotulinumtoxinA is an
effective and well-tolerated formulation of
BoNTA. In clinical trials, incobotulinumtox-
inA displays comparable efficacy and safety
to onabotulinumtoxinA and abobotulinum-
toxinA when administered at dose conversion
ratios of 1 : 1 and 1 : 3, respectively. It is not
only free from complexing proteins, but also
inactivated or denatured neurotoxin, bring-
ing the foreign protein load to a minimum.
In this respect, incobotulinumtoxinA repre-
sents a novel advance in BoNTA formula-
tions that have now been used in aesthetic
applications for over 20 years. This develop-
ment has yielded a product that is also sta-
ble at room temperature for long-term stor-
age, giving it a practical advantage. As the

6 IncobotulinumtoxinA (Xeomin®/Bocouture®)

(a)
(c)
(b)
0.33 U
0.5 U
3 U
Figure .Microinjection technique around the eye. (c) Taken 2 weeks after (a) and (b) at maximum eyebrow
elevation. Note smoothing of periorbital wrinkling above eyebrow and at lower lid without losing significant
eyebrow height.
use of BoNTA in cosmetic medicine contin-
ues to grow and long-term comparative stud-
ies are published, evidence will accumulate to
resolve the controversial issue of complexing
proteins and their role in antibody-induced
secondary treatment failure.
References
Carruthers A, Carruthers J. Botulinum
toxin products overview. Skin Ther Lett
2008;13:1–4.
Bigalke H. Properties of pharmaceutical
products of botulinum neurotoxins. In
Jankovic J, Albane A, Atassi MZ, et al., eds.
Botulinum Toxin. Philadelphia: Saunders;
2009:389–397.
Frevert J. Content of botulinum neurotoxin
in Botox®/Vistabel®, Dysport®/Azzalure®,
and Xeomin®/Bocouture®. Drugs R D
2010;10:67–73.
Benecke R, Jost WH, Kanovsky P, et al. A
new botulinum toxin type A free of
complexing proteins for treatment of
cervical dystonia. Neurology 2005;64:
1949–1951.
Roggenk¨amper P, Jost WH, Bihari K, et al.
Efficacy and safety of a new botulinum
toxin type A free of complexing proteins in
the treatment of blepharospasm. J Neural
Transm 2006;113:303–312.
Sattler G, Callander M, Grablowitz D, et al.
Non-inferiority of NT201, free from