Quy Trình Tiêm Botulinum Toxin Trong Da Liễu Thẩm Mỹ (Cosmetic Dermatology Series)

33
5 
Basic science: Abobotulinum toxin A
Eisele KH, Fink K, Vey M, et al 2011 Studies on the dissociation
of botulinum neurotoxin type A complexes. Toxicon
57(4):555–565
Hambleton P, Pickett AM 1994 Potency equivalence of botulinum
toxin preparations. Journal of the Royal Society of Medicine
87(11):719
Hexsel D, Dal’Forno T, Hexsel C, et al 2008 A randomized
pilot study comparing the action halos of two commercial
preparations of botulinum toxin type A. Dermatologic Surgery
34:52–59
Inoue K, Fujinaga Y, Watanabe T, et al 1996 Molecular composition
of Clostridium botulinum type A progenitor toxins. Infection
and Immunity 64(5):1589–1594
Kane MA, Brandt F, Rohrich RJ, et al 2009 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. Plastic and
Reconstructive Surgery 124(5):1619–1629
Karsai S, Adrian R, Hammes S, et al 2007 A randomized double-
blind study of the effect of botox and dysport/reloxin on
forehead wrinkles and electromyographic activity. Archives of
Dermatology 143(11):1447–1449
Kessler KR, Skutta M, Benecke R 1999 Long-term treatment of
cervical dystonia with botulinum toxin A: efficacy, safety, and
antibody frequency. German Dystonia Study Group. Journal of
Neurology 246(4):265–274
Kerscher M, Roll S, Becker A, et al 2012 Comparison of the spread
of three botulinum toxin type A preparations. Archives of
Dermatological Research 304(2):155–161
Koriazova LK, Montal M 2003 Translocation of botulinum
neurotoxin light chain protease through the heavy chain
channel. Nature Structural Biology 10(1):13–18
Lawrence I, Moy R 2009 An evaluation of neutralizing antibody
induction during treatment of glabellar lines with a new US
formulation of botulinum neurotoxin type A. Aesthetic Surgery
Journal 29(6):S66–S71
Lee S-K 2007 Antibody-induced failure of botulinum toxin type A
therapy in a patient with masseteric hypertrophy. Dermatologic
Surgery 33(suppl 1):S105–S110
Lietzow MA, Gielow ET, Le D, et al 2008 Subunit stoichiometry
of the Clostridium botulinum type A neurotoxin complex
determined using denaturing capillary electrophoresis. Protein
Journal 27(7-8):420–425
Lowe P, Patnaik R, Lowe N 2006 Comparison of two
formulations of botulinum toxin type A for the treatment
of glabellar lines: A double-blind, randomized study.
Journal of the American Academy of Dermatology 55(6):
975–980
Monheit G, Carruthers A, Brandt F, et al 2007 A randomized,
double blind, placebo controlled study of botulinum toxin A for
the treatment of glabellar lines: Determination of optimal dose.
Dermatologic Surgery 33:51–59
Monheit G, Cohen J, Reloxin Investigational Group 2009
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. Journal of the American Academy of Dermatology
61(3):421–425
Nestor MS, Ablon GR 2011 Comparing the clinical attributes of
abobotulinumtoxinA and onabotulinumtoxinA utilizing a novel
contralateral frontalis model and the frontalis activity
measurement standard. Journal of Drugs in Dermatology
10(10):1148–1157
Nettar KD, Yu KC, Bapna S, et al 2011 An internally controlled,
double-blind comparison of the efficacy of onabotulinumtoxinA
and abobotulinumtoxinA. Archives of Facial Plastic Surgery
13(6):380–386
As aesthetic physicians become familiar with this
second botulinum toxin product in the USA, new innova­
tions and techniques will evolve and new data beyond the
clinical trials will emerge. It is said the real clinical trial
for a drug or device occurs in the first 2 years after release.
With the large usage numbers, we gain a further and more
detailed understanding of both efficacy and safety for this
distinct product.
Further reading
Alam M, Dover JS, Arndt KA 2002 Pain associated with injection
of botulinum a exotoxin reconstituted using isotonic sodium
chloride with and without preservative: a double-blind,
randomized controlled trial. Archives of Dermatology
138(4):510–514
Allen SB, Goldenberg NA 2012 Pain difference associated with
injection of abobotulinumtoxina reconstituted with preserved
saline and preservative-free saline: a prospective, randomized,
side-by-side, double-blind study. Dermatologic Surgery Jan 23;
doi: 10.1111/j.1524-4725.2011.02284.x. [Epub ahead of print]
Allergan, Inc. 2010 Safety analysis (data on file). Online. Available:

clinical_information.aspx, 10 January 2010
Ascher B, Talarico S, Cassuto D, et al 2010 International consensus
recommendations on the aesthetic usage of botulinum toxin
type A (Speywood Unit) – part I: upper facial wrinkles. Journal
of the European Academy of Dermatology and Venereology
24:1278–1284
Ascher B, Talarico S, Cassuto D, et al 2010 International consensus
recommendations on the aesthetic usage of botulinum toxin
type A (Speywood Unit) – Part II: Wrinkles on the middle and
lower face, neck and chest. Journal of the European Academy of
Dermatology and Venereology 24(11):1285–1295
Ascher B, Rzany BJ, Grover R 2009 Efficacy and safety of
botulinum toxin type A in the treatment of lateral crow’s feet:
double-blind, placebo-controlled, dose-ranging study.
Dermatologic Surgery 35:1478–1486
Ascher B, Zakine B, Kestemont P, et al 2004 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. Journal of the American Academy
of Dermatology 51(2):223–233
Carruthers A, Carruthers J, Lowe NJ, et al for the BOTOX
Glabellar Lines I & II Study Groups 2004 One-year,
randomized, multicenter, two-period study of the safety and
efficacy of repeated treatments with botulinum toxin type A in
patients with glabellar lines. Journal of Clinical Research 7:1–20
Carruthers J, Lowe NJ, Mentor MA, et al 2003 Double-blind
placebo-controlled study of the safety and efficacy of Botulinum
toxin type A for patients with glabellar lines. Plastic and
Reconstructive Surgery 112:1089–1098
Carruthers J, Fagien S, Matarasso SL 2004 Consensus
recommendations on the use of botulinum toxin type A in
facial aesthetics. Plastic and Reconstructive Surgery 114
(suppl 1):S1–S22
Dressler D, Wohlfahrt K, Meyer-Rogge E, et al 2010 Antibody-
induced failure of botulinum toxin A therapy in cosmetic
indications. Dermatologic Surgery 36(s4):2182–2187
Eisele KH 2009 Is there a role for complexing proteins in
pharmaceutical neurotoxin formulations? Presented at the
International Masters Course on Aging Skin, 8-11 January 2009,
Paris, France
Eisele KH, Taylor HV 2008 Dissociation of the 900 kDa neurotoxin
complex from C. botulinum under physiological conditions.
Toxicon 51(suppl 1):10

34
Botulinum toxin
Schiavo G, Matteoli M, Montecucco C 2000 Neurotoxins affecting
neuroexocytosis. Physiological Reviews 80(2):717–766
Trindade de Almeida AR, Marques E, de Almeida J, et al 2007
Pilot study comparing the diffusion of two formulations of
botulinum toxin type A in patients with forehead hyperhidrosis.
Dermatologic Surgery 33(1 spec. no.):S37–S43
Vandenbergh PYK, Lison DF 1998 Dose standardization of
botulinum toxin. Advances in Neurology 78:231–235
Wohlfarth K, Schwandt I, Wegner F, et al 2008 Biological activity
of two botulinum toxin type A complexes (Dysport(r) and
Botox(r)) in volunteers: a double-blind, randomized, dose-
ranging study. Journal of Neurology 255(12):1932–1939
Pickett A 2009 BoNT-A: myths and realities. Presented at the
International Masters Course on Aging Skin, 8-11 January 2009,
Paris, France
Pickett A 2009 Dysport(r): Pharmacological properties and factors
that influence toxin action. Toxicon 54(5):683–689
Rubin M, Dover J, Glogau R, et al 2009 The efficacy and safety
of a new US botulinum toxin type A in the retreatment of
glabellar lines following open-label treatment. Journal of Drugs
in Dermatology 8(5):439–444
Rzany B, Ascher B, Monheit G 2010 Treatment of glabellar lines
with botulinum toxin type A (Speywood Unit): a clinical
overview. Journal of the European Academy of Dermatology and
Venereology 24 (suppl 1):1–14
Sampaio C, Costa J, Ferreira JJ 2004 Clinical comparability of
marketed formulations of botulinum toxin. Movement Disorders
19(suppl 8):S129–S136

the field of aesthetic medicine. IncobotulinumtoxinA is
licensed in Germany, the UK, and all other major Euro­
pean countries under the trade name Bocouture® for the
treatment of moderate to severe glabellar frown lines in
adults below 65 years when the severity of these lines has
an important psychological impact for the patient, as well
as Russia, Mexico and Argentina, and South Korea for the
treatment of hyperkinetic facial lines. It was recently
approved by the Food and Drug Administration (FDA) in
2011 for the treatment of glabellar frown lines in the
USA, where it is marketed as Xeomin®. Incobotulinum­
toxinA differs from the other botulinum toxin type A
products on the market (onabotulinumtoxinA, Botox® / Vis­
tabel®, Allergan, Irvine, CA, USA; abobotulinumtoxinA,
Dysport® / Azzalure®, Ipsen Ltd, Berkshire, UK) – which
contain other clostridial proteins (complexing proteins,
also named neurotoxin-associated proteins) – because it
contains only the 150 kDa protein responsible for the
therapeutic effect.
Properties of incobotulinumtoxinA
Purity
Botulinum toxin is produced by anaerobic fermentation
of the bacterium Clostridium botulinum type A (Hall
strain). During the manufacture of incobotulinumtoxinA,
following fermentation and extraction of the toxin, the
complexing proteins are removed by chromatography.
Thus incobotulinumtoxinA contains markedly less clos­
tridial protein than do onabotulinumtoxinA and abobotu­
linumtoxinA (Fig. 6.1). In addition, incobotulinumtoxinA
has the highest specific biological activity of all three prod­
ucts. A high-sensitivity sandwich ELISA was used in a
study by Frevert to measure the amount of botulinum
toxin type A per 100 U of onabotulinumtoxinA, incobotu­
linumtoxinA and abobotulinumtoxinA. The results were
0.73 ng, 0.44 ng and 0.65 ng, respectively, giving incobotu­
linumtoxinA the highest specific biological activity (U/ng
neurotoxin) at 227 U/ng compared with 137 U/ng for
onabotulinumtoxinA and 154 U/ng for abobotulinumtox­
inA (but it must be noted that the units of abobotulinum­
toxinA are different from those of onabotulinumtoxinA
and incobotulinumtoxinA). This suggests that, in addition
to containing complexing proteins, onabotulinumtoxinA
may also contain denatured / inactivated neurotoxin, unlike
6 
Basic science: Xeomin®
Jürgen Frevert, Gerhard Sattler
Introduction
IncobotulinumtoxinA (NT 201 / Xeomin®) is manufac­
tured by Merz Pharmaceuticals GmbH (Frankfurt,
Germany). It is used to treat certain neurological condi­
tions (cervical dystonia, blepharospasm, and post-stroke
upper limb spasticity). In addition, it has various uses in
Summary and Key Features
•
IncobotulinumtoxinA (NT 201, Xeomin®,
Bocouture®) is an effective and well-tolerated
treatment for facial wrinkles and is approved for
the treatment of glabellar frown lines in many
countries
•
IncobotulinumtoxinA differs from other
commercially available botulinum toxin type A
products as it is free from complexing proteins and
other impurities
•
Complexing proteins have no therapeutic effect
and do not prevent the active neurotoxin from
diffusing and may lead to an increased risk of
antibody formation
•
IncobotulinumtoxinA is highly stable and can be
stored at room temperature
•
IncobotulinumtoxinA has a higher specific
biological activity than other available botulinum
toxin type A preparations, such as
onabotulinumtoxinA (Botox® / Vistabel) or
abobotulinumtoxinA (Dysport® / Azzalure®)
•
In practice, the clinical equipotency of
incobotulinumtoxinA and onabotulinumtoxinA has
been proven when a dose conversion ratio of 1 : 1
is used. Multiple small dose injections result in a
precise and predictable outcome
•
The best results are achieved when the face is
treated as a whole, since treating some muscles
or areas in isolation can draw attention to the
untreated areas

36
Botulinum toxin
incobotulinumtoxinA (see Fig. 6.1). Like the other botuli­
num toxin type A products, incobotulinumtoxinA contains
human serum albumin as an excipient but, whereas inco­
botulinumtoxinA contains sucrose, abobotulinumtoxinA
contains lactose, and onabotulinumtoxinA contains neither
of these sugars (Table 6.1).
Stability
It was assumed that complexing proteins are required to
stabilize the neurotoxin, but incobotulinumtoxinA can be
stored at room temperature and without refrigeration
for 4 years. In addition to the practical advantage of not
requiring cold storage, incobotulinumtoxinA did not lose
activity in a temperature stress stability study by Grein &
Fink. Furthermore, its quality did not decrease following
storage at 60°C for 1 month. In accordance with the pre­
scribing information, once reconstituted, incobotulinum­
toxinA should be stored at 2–8°C and used within 24
hours to limit the risk of bacterial growth.
Figure 6.1  The amount of clostridial protein and neurotoxin
protein contained in incobotulinumtoxinA, onabotulinumtoxinA and
abobotulinumtoxinA per dose approved for the treatment of glabellar
frown lines.
1
0.8
0.6
0.4
0.2
1.2
Clostridial protein
Neurotoxin protein
0
OnabotulinumtoxinA
IncobotulinumtoxinA
AbobotulinumtoxinA
Protein applied (ng)
Pearl 1 
The high stability of incobotulinumtoxinA allows it to be
stored at room temperature.
Diffusion
It is important to understand the difference between
diffusion and spread of the neurotoxin. Diffusion is the
passive movement of the toxin down a concentration gra­
dient within the medium. In contrast, spread occurs when
the injected molecule travels from the site of injection.
The latter is usually a result of the injection technique,
skills of the injector, injected volume, and needle size and
the consistency of the treated tissue and can cause unde­
sirable effects in adjacent muscles.
Under acidic conditions the complexing proteins form
a high-molecular-weight complex (900 kDa) with the
neurotoxin. Accordingly, it was speculated that complex­
ing proteins limit the diffusion of the active neurotoxin
within the target muscle. However, experiments in
animals and humans suggest otherwise. The diffusion of
onabotulinumtoxinA, abobotulinumtoxinA, and incobotu­
linumtoxinA was investigated by Carli and colleagues in a
mouse model using staining for neural cell adhesion mol­
ecule (N-CAM) as a marker. N-CAM is expressed only
by denervated muscle cells and so is suitable for testing
Table 6.1  Botulinum toxin type A product comparison*
OnabotulinumtoxinA
AbobotulinumtoxinA
IncobotulinumtoxinA
Units per vial
50 U
125 U
50 U
Clostridial strain
Hall
Hall
Hall
Type A
Type A
Type A
Molecular composition
150 kDa neurotoxin and
complexing proteins
150 kDa neurotoxin and
compexing proteins
150 kDa neurotoxin
Recommended dose for treatment of glabellar
lines in LD50 units
20 U
50 U
20 U
Excipients
HSA
HSA
HSA
Sodium chloride
Lactose monohydrate
Sucrose
HSA, human serum albumin.
*(Bocouture® 2010 Summary of Product Characteristics. Merz Pharmaceuticals GmbH, Germany; Vistabel® 2008 Summaryof Product
Characteristics. Allergan, Inc, Irvine, CA, USA; Azzalure® 2010, Galderma Ltd, Hertfordshire, UK.)

37
6 
Basic science: Xeomin®
treatment did not induce the production of neutralizing
antibodies. This suggests that the presence of complexing
proteins might be clinically relevant.
Clinical performance of incobotulinumtoxinA
Introduction
IncobotulinumtoxinA has been assessed as a neurological
and aesthetic treatment in a robust preclinical and clinical
development program. In an open label, intraindividual,
controlled study by Jost and colleagues in healthy male
volunteers, incobotulinumtoxinA was compared with ona­
botulinumtoxinA in terms of its ability to reduce the
compound muscle action potential in the extensor digito­
rum brevis (EDB) muscle of the foot at various timepoints
post-injection. There were no significant differences in the
degree of paralysis at a 1 : 1 dose ratio. In a similar study
by Wohlfarth and colleagues in healthy volunteers injected
in the EDB muscle with a range of doses of incobotu­
linumtoxinA compared with equal doses of onabotulinum­
toxinA, there was no difference in the efficacy of these
products and neither treatment diffused into adjacent
muscles.
Neurological indications
IncobotulinumtoxinA is indicated for the treatment of
three neurological conditions: the symptomatic treatment
of blepharospasm, cervical dystonia of a predominantly
rotational form (spasmodic torticollis), and post-stroke
spasticity of the upper limb presenting with flexed wrist
and clenched fist in adults.
A large, prospective, randomized, double-blind trial by
Benecke and colleagues demonstrated that incobotu­
linumtoxinA was not inferior to onabotulinumtoxinA for
the treatment of cervical dystonia. In this study, 463
patients received intramuscular injections of 70–300 U of
incobotulinumtoxinA or onabotulinumtoxinA and, in both
groups, the severity score on the Toronto Western Spas­
modic Torticollis Scale (TWSTRS) improved by 11 points
by day 28. For the treatment of blepharospasm, in a large,
multi-center double-blind Phase III study by Roggenkäm­
per and co-workers, 256 patients received a similar mean
total dose of either incobotulinumtoxinA or onabotu­
linumtoxinA (39.6 U and 40.8 U, respectively). Three
weeks after injection, patients in both groups showed a
significant improvement in the symptoms of blepharo­
spasm, indicating that both products were effective treat­
ments and the non-inferiority of incobotulinumtoxinA to
onabotulinumtoxinA was confirmed.
In a randomized, double-blind, placebo-controlled,
Phase III study by Kanovsky and colleagues, 148 patients
with an Ashworth Scale score of at least two for wrist and
finger flexors following stroke were randomly assigned to
receive incobotulinumtoxinA or placebo. The median dose
of incobotulinumtoxinA administered was 320 U. Four
weeks after treatment, a significantly larger proportion of
for muscles affected by botulinum toxin type A. Results
indicated that, following injection into the tibialis anterior
(TA) muscle of the mouse leg, there was limited diffusion
into tissue that was adjacent to the site of injection and
no difference in diffusion was observed between incobotu­
linumtoxinA, onabotulinumtoxinA, and abobotulinum­
toxinA using a 1 : 1 : 4 dose conversion ratio.
A recent clinical study by Kerscher and colleagues
showed no difference in the size of the anhidrotic area
produced following injection of 5 U of incobotulinum­
toxinA (free from complexing proteins) compared with
5 U of onabotulinumtoxinA (containing complexing pro­
teins) on either side of the forehead after 6 weeks and
6 months.
These results suggest that complexing proteins have no
effect on diffusion. The reason for this was revealed by a
recent study by Eisele and co-workers on the stability of
the complexes and the kinetics of their dissociation. This
report showed that the high molecular weight complexes
presumably present in onabotulinumtoxinA and abobotu­
linumtoxinA are dissociated by reconstitution and physi­
ological pH. Reconstitution of the commercially available
complexing-protein-containing products, onabotulinum­
toxinA and abobotulinumtoxinA, with saline (as in the
clinic) led to a complete dissociation of the 900 kDa
complexes and resulted in at least 85% of the neurotoxin
being in its free form. In addition, it was shown that more
than 80% of the 150 kDa neurotoxin was released from
complexes in under a minute at physiological pH. There­
fore, following injection into target tissue, where the pH
is usually close to neutral, any remaining complexes dis­
sociate immediately, making the presence or absence of
complexing proteins in the original preparation irrelevant
in terms of diffusion. Complexing proteins do not contrib­
ute to the therapeutic effect and are therefore only clos­
tridial impurities.
Pearl 2 
Complexing proteins have no function in limiting the diffusion
of the neurotoxin. IncobotulinumtoxinA, with its 150 kDa
neurotoxin, is efficacious without any other clostridial proteins.
Immunogenicity
Botulinum toxin is a foreign protein and is therefore anti­
genic. Immunogenicity remains a controversial subject in
the field of botulinum toxin type A treatment in aesthet­
ics. While the generation of neutralizing antibodies is more
common in therapeutic indications, where dosages tend
to be much larger, cases of secondary non-responsiveness
have been reported in aesthetics. In a study by Blümel and
colleagues investigating the generation of neutralizing anti­
bodies following repeated injections of onabotulinum­
toxinA, abobotulinumtoxinA or incobotulinumtoxinA into
New Zealand white rabbits, neutralizing antibodies were
detected after both abobotulinumtoxinA and onabotu­
linumtoxinA treatment. In contrast, incobotulinumtoxinA

38
Botulinum toxin
defined as a patient with a greater than two-point improve­
ment in glabellar frown lines on a four-point scale, as
assessed by both investigator and patient at day 30 at
maximum frown. In both studies, the responder rate with
incobotulinumtoxinA was superior to that with placebo
(p < 0.0001). In addition, when a responder was defined
as a patient with a score of 0 (none) or 1 (mild wrinkles)
at day 30 post-injection on the FWS, as assessed by the
investigator, 79.9% and 76.4% of patients respectively
were responders at maximum frown (p < 0.0001 com­
pared with placebo in both trials) and responder rates
were also superior to placebo (p < 0.0001) at rest (66.8%
and 86.3% respectively; Merz Pharmaceuticals GmbH,
data on file).
In a further recent clinical trial of incobotulinumtoxinA
for the treatment of glabellar frown lines, the efficacy
and safety of repeated dosing were studied. Responder
rates remained high, in the range of 79–90% at maximum
frown, as assessed by the investigator 30 days after
treatment in each cycle over a maximum 2-year period
at maximum frown, where responders were defined as
patients with a score of 0 or 1 on the FWS (Merz Phar­
maceuticals GmbH).
IncobotulinumtoxinA has also demonstrated efficacy in
the off-label treatment of periorbital lines. In a double-
blind, randomized, proof-of-concept study by Prager and
colleagues, the clinical efficacy of incobotulinumtoxinA
was compared with onabotulinumtoxinA in a 1 : 1 dose
ratio. Twelve units of each product were compared in
an intraindividual, split-face study. One month post-
treatment, response rates were 95% for incobotulinum­
toxinA compared with 90% for onabotulinumtoxinA,
although this difference was not statistically significant.
The response rates after 3 and 4 months were 89% and
84% respectively, for both products. These results dem­
onstrate that there was no difference in the efficacy of
incobotulinumtoxinA and onabotulinumtoxinA for the
treatment of periorbital lines.
Another double-blind, randomized pilot study by the
same team compared the efficacy of incobotulinumtoxinA
with abobotulinumtoxinA in an intraindividual, split-face
design for the treatment of periorbital lines over a period
of 4 months. The results showed no significant difference
in efficacy between incobotulinumtoxinA and abobotu­
linumtoxinA at the dose ratio of 1 : 3 used in this study.
Safety in aesthetics
In the head-to-head study by Sattler et al comparing inco­
botulinumtoxinA with onabotulinumtoxinA for the treat­
ment of glabellar frown lines, the incidence of adverse
events (AEs) was low. Treatment-emergent AEs that were
considered to be related to study treatment occurred in
3.2% of patients in the incobotulinumtoxinA group and
5.2% of patients in the onabotulinumtoxinA group. The
most commonly reported AE was headache. In Imhof &
Kuhne’s Phase III trial of incobotulinumtoxinAs for glabel­
lar frown lines, four patients (3.8%) experienced TEAEs
incobotulinumtoxinA-treated patients had shown an
improvement of at least one point on the five-point Ash­
worth Scale in wrist flexors compared with placebo-
treated patients. IncobotulinumtoxinA treatment led to
statistically significant improvements in muscle tone and
disability in patients with post-stroke upper limb spastic­
ity compared with placebo, confirming incobotulinum­
toxinA as an effective treatment.
Safety summary in neurological conditions
In all three Phase III clinical trials in neurological indica­
tions, incobotulinumtoxinA was well tolerated and most
adverse events were of mild or moderate intensity. When
compared with onabotulinumtoxinA using a 1 : 1 dose
ratio, the safety and tolerability profiles of both products
were similar.
Aesthetic indications
IncobotulinumtoxinA has been studied by Sattler and col­
leagues in a large Phase III prospective, multicenter, ran­
domized, rater- and patient-blind clinical trial designed to
investigate the non-inferiority of incobotulinumtoxinA
compared with onabotulinumtoxinA for the treatment of
glabellar frown lines. A total of 381 women with moderate
to severe glabellar frown lines at maximum frown (severity
score of 2 or 3 on the four-point Facial Wrinkle Scale
[FWS]) were randomized in a 3 : 1 ratio (incobotulinum­
toxinA : onabotulinumtoxinA) to receive 24 U of either
product. Four weeks after injection, response rates at
maximum frown were assessed by independent raters and
were found to be 96.4% in the incobotulinumtoxinA group
and 95.7% in the onabotulinumtoxinA group. Twelve
weeks after injection, the response rates at maximum
frown remained high at 80.1% and 78.5% for incobotu­
linumtoxinA and onabotulinumtoxinA respectively. A
responder was defined as a patient with at least a one-point
improvement on the FWS. Statistical analysis confirmed
that incobotulinumtoxinA was not inferior to onabotu­
linumtoxinA for the treatment of glabellar frown lines.
In another prospective, open-label, multicenter Phase
III clinical trial by Imhof & Kuhne, 105 patients received
one cycle of 20 U of incobotulinumtoxinA for the treat­
ment of moderate to severe glabellar frown lines. A
responder was defined as a patient with an improvement
of at least one point on the FWS and the response rate at
maximum frown was 98.1% at day 28 and remained high
at 80.0% on day 84, as assessed by the investigator dem­
onstrating the efficacy of incobotulinumtoxinA in the
treatment of glabellar frown lines.
Two recent large, randomized, double-blind, placebo-
controlled Phase III trials of incobotulinumtoxinA in
the treatment of glabellar frown lines confirmed the effi­
cacy and tolerability previously demonstrated in this indi­
cation (Merz Pharmaceuticals GmbH, data on file). In
addition, these trials used new, more stringent definitions
of responders, mandated by the FDA. A responder was

39
6 
Basic science: Xeomin®
A physician should palpate patients’ facial muscles and
ask them to activate certain muscles so that the physician
can feel the characteristics of muscle action as a clinical
control. For teaching purposes a single muscle may be
analyzed and described but, in clinical practice, whole
areas are treated and a muscle cannot be considered in
isolation. It is important to consider the action of pairs of
agonist and antagonist muscles. For example, treating the
orbicularis weakens the antagonistic action experienced by
the frontalis and results in brow elevation, and a deep
injection into the caudal part of the frontalis will trigger
the proximal fibers of the frontalis for enhanced results of
brow elevation.
that were deemed related to study treatment: three head­
aches and one case of worsening of pre-existing carotid
artery stenosis. In the study of crow’s feet by Prager and
co-workers, the majority of patients (>68%) rated toler­
ability as very good over the whole study period, according
to a subjective five-point scale.
Summary
Figure 6.2  Dynamic musculature of the face: (A) superficial, (B) intermediate, (C) deep.
Levator
labii superioris
Mentalis
Levator
anguli oris
Corrugator
Platysma
Depressor
labii inferioris
Orbicularis
oris
Frontalis
Procerus
Depressor
anguli oris
Orbicularis
oculi
C
B
A
Pearl 4 
A physician should begin a consultation by asking the patient
what he / she hopes to achieve from treatment, and should
conduct a full patient evaluation.
Pearl 3 
Results from clinical trials have demonstrated that incobotu­
linumtoxinA has equivalent efficacy and similar tolerability to
onabotulinumtoxinA when used in a 1 : 1 dose conversion
ratio.
This is reflected by the fact that, in clinical practice, inco­
botulinumtoxinA and onabotulinumtoxinA are treated as
equivalent in terms of dosage and the approved dose for
glabellar frown lines is 20 U for both products.
Clinical experience with incobotulinumtoxinA
The clinical development program for incobotulinumtox­
inA has demonstrated excellent efficacy in the treatment
of glabellar frown lines and periorbital lines, and it is well
tolerated. In addition, the characteristics of incobotu­
linumtoxinA: the lack of complexing proteins, high spe­
cific biological activity, predictability of results, and good
tolerability, recommend it as a precise and effective tool
in the field of aesthetic medicine. When making the deci­
sion of which product to use, physicians have to consider
factors such as therapeutic efficacy, patient satisfaction,
safety, price, and handling. IncobotulinumtoxinA has
shown comparable efficacy, patient satisfaction, and safety
to onabotulinumtoxinA using equal doses, and has the
benefit of having no need for cold storage. Along with the
emergence of such an efficient product, injection tech­
niques have improved over time so that treatment has
become more sophisticated, with more subtle results and
avoidance of the ‘frozen’ look. With multiple small doses
and a sound awareness of facial anatomy (Fig. 6.2), inco­
botulinumtoxinA gives very predictable results.
Pearl 5 
It is important that the face be considered as a whole to avoid
drawing undue attention to untreated regions. A total face
approach is recommended.
In addition, accurate injection of small doses should
avoid any undesirable effects due to spread of the neuro­
toxin. This is crucial in the face, where muscles are small.
If the wrong part of a muscle is targeted when performing
a ‘brow lift’, brow ptosis can occur leaving the patient
looking tired. Incorrect treatment of lateral canthal lines
can have a detrimental effect on smiling, for example.
The total face approach with
incobotulinumtoxinA
To obtain the most effective improvement in facial wrin­
kles, treatment of the whole face, as described below, is
recommended. The glabellar region can display single and
double vertical lines and can be treated with one or two
injections into the superior region of the procerus along
the median line (total dose of 4–6 U), along with further
medial injections into the central part of the muscle (total

40
Botulinum toxin
Figure 6.3  The total face approach: injection sites to treat the glabellar region (A), eyebrows (B), mephisto (C), lateral canthal lines (D),
forehead (E), lower eyelid (F), open eye (G), bunny lines (H), gummy smile (I), upper and lower lip (J), marionette lines (K), mentalis (L), and
platysma (M). Blue dots, intramuscular injections of 1–2 U depending on the clinical situation and gender. Light blue circles, intradermal
injections of 0.5–1 U. Reproduced from Sattler G, et al 2009 Bildatlas der ästhetischen Botulinumtoxin-Therapie. KVM Verlag with permission
from KVM Verlag.
A
B
C
D
G
F
E
H
L
M
I
J
K

41
6 
Basic science: Xeomin®
dose of 2–4 U). In addition, treatment should include one
medial injection (1–2 U) in the direction of the fibers of
the corrugator supercilii and one superficial injection into
the medial aspect of the orbicularis oculi (2–4 U; Fig.
6.3A). Treatment of the eyebrow includes four injections
in the part of the eyebrow above the orbital rim: super­
ficial injections in fibers of the orbicularis and deep
injections in caudal fibers of the frontalis (Fig. 6.3B). The
mephisto appearance should be treated at the point of
maximum contraction, with 1 U incobotulinumtoxinA
injected into the lateral part of the frontalis (Fig. 6.3C).
For lateral canthal lines, lateral intracutaneous injections
and deep subcutaneous injections above the lateral orbital
rim (1–2 U per injection) can be performed into the orbi­
cularis oculi (Fig. 6.3D). There may be no need to treat
the center of the frontalis as this can enhance brow ptosis
and a ‘tired’ look. Horizontal forehead lines form part of
normal expressions of surprise and interest and hence do
not necessarily have negative connotations. It is therefore
advisable to delay treatment of the frontalis until approxi­
mately 4 weeks after treatment of the glabellar region if
it is still deemed necessary. The forehead can be treated
by five deep muscle injections of 1–2 U each administered
in a V-shape configuration in the medial area of the fron­
talis and multiple superficial injections of (0.5–1 U lateral
to the midline (Fig. 6.3E)).
In cases of fine wrinkles in the area of the lower eyelid
four intradermal injections of 0.5 U incobotulinumtoxinA,
in a volume of approximately 0.04 mL (i.e. a more diluted
preparation of incobotulinumtoxinA), can be used treat
each lower eyelid. In order to open the eye, a single super­
ficial pretarsal injection of 1–2 U in the orbicularis oculi
along the mid-pupillary line can also be administered (Fig.
6.3F, G). Treatment of bunny lines can be achieved by one
or two superficial injections of 1–2 U in the medial, supe­
rior part of the nasal wing (Fig. 6.3H). A single deep injec­
tion of 1 U in part of the levator labii superioris at the base
of the nasolabial fold can treat a ‘gummy’ smile (Fig. 6.3I).
An injection of 0.5 U between the vermilion border and
the white portion of the lip can improve the appearance
of the upper and lower lips (Fig. 6.3J), and a deep injection
(1–2 U) lateral to the marionette line will cover the supe­
rior parts of the platysma muscle and the depressor anguli
oris to treat marionette lines. Care should be taken to
ensure that the distance to the corner of the mouth is at
least 1 cm in the inferior direction to prevent diffusion into
the orbicularis oris (Fig. 6.3K). To treat the chin, both the
left and right mentalis muscles should be injected. This
should preferably be done at the top of the corner of the
chin in the direction of the neck to prevent treatment of
the orbicularis oris (Fig. 6.3L). Finally the platysma muscle
can be treated by multiple injections of 1–2 U along the
hypertrophic bands, 2–3 cm apart (Fig. 6.3M).
Conclusion
Botulinum toxins have been used for aesthetic purposes
for almost 25 years and are an effective treatment of
facial wrinkles. According to the cosmetic surgery national
data bank statistics of the American Society for Aesthetic
Plastic Surgery (found at ), botulinum
toxin treatment was the most common non-surgical aes­
thetic procedure performed in the USA in 2010. Inco­
botulinumtoxinA (NT 201, Xeomin®, Bocouture®) is a
highly purified botulinum toxin type A that is free from
complexing proteins, potent and well tolerated, with
proven efficacy in neurological and aesthetic indications.
Studies have shown that incobotulinumtoxinA is clinically
equipotent to onabotulinumtoxinA in the treatment of
cervical dystonia, blepharospasm, and glabellar frown
lines and these two products are routinely used in a 1 : 1
dose conversion ratio. With accurate injection of small
doses of incobotulinumtoxinA, unwanted effects can be
avoided, leaving the patient with a predictable, subtle and
softer look.
Further reading
[anon] 2010 Azzalure®. Summary of Product Characteristics. Ipsen
Ltd, Berkshire, UK
[anon] 2010 Bocouture®. Summary of Product Characteristics.
Merz Pharmaceuticals GmbH, Germany
[anon] 2008 Vistabel®. Summary of Product Characteristics.
Allergan, Inc, Irvine, CA, USA
[anon] 2011 Xeomin®. Summary of Product Characteristics. Merz
Pharmaceuticals GmbH, Germany
Benecke R, Jost WH, Kanovsky P, et al 2005 A new botulinum
toxin type A free of complexing proteins for treatment of
cervical dystonia. Neurology 64:1949–1951
Blümel J, Frevert J, Schwaier A 2006 Comparative antigenicity of
three preparations on boutlinum neurotoxin A in the rabbit.
Neurotoxicity Research 9:238
Borodic G 2006 Immunologic resistance after repeated botulinum
toxin type A injections for facial rhytides. Ophthalmic Plastic
and Reconstructive Surgery 22:239–240
Carli L, Montecucco C, Rossetto O 2009 Assay of diffusion of
different botulinum neurotoxin type A formulations injected in
the mouse leg. Muscle Nerve 40:374–380
Carruthers A, Carruthers J 2008 Botulinum toxin products
overview. Skin Therapy Letter 2013:1–4
Dressler D, Adib Saberi F 2007 New formulation of Botox:
complete antibody-induced treatment failure in cervical
dystonia. Journal of Neurology, Neurosurgery, and Psychiatry
78:108–109
Dressler D, Wohlfahrt K, Meyer-Rogge E, et al 2010
Antibody-induced failure of botulinum toxin a therapy in
cosmetic indications. Dermatologic Surgery 36(suppl 4):
2182–2187
Eisele K-H, Fink K, Vey M, et al 2011 Studies on the dissociation
of botulinum neurotoxin type A complexes. Toxicon 57:555–
565
Frevert J 2010 Content of botulinum neurotoxin in
Botox® / Vistabel®, Dysport® / Azzalure®, and
Xeomin® / Bocouture®. Drugs R D 2010;10:67–73
Grein S, Mander GJ, Fink K 2011 Stability of botulinum
neurotoxin type A, devoid of complexing proteins. Botulinum
Journal 2:49–58
Imhof M, Kuhne U 2011 A Phase III study of incobotulinumtoxinA
in the treatment of glabellar frown lines. Journal of Clinical and
Aesthetic Dermatology 4:28–34
Jost WH, Kohl A, Brinkmann S, et al 2005 Efficacy and tolerability
of a botulinum toxin type A free of complexing proteins

42
Botulinum toxin
A split-face, double-blind, proof-of-concept study. Dermatologic
Surgery 36:2155–2160
Roggenkämper P, Jost WH, Bihari K, et al 2006 Efficacy and safety
of a new botulinum toxin type A free of complexing proteins in
the treatment of blepharospasm. Journal of Neural Transmission
113:303–312
Sattler G, Callander M, Grablowitz D, et al 2010 Noninferiority of
incobotulinumtoxinA, free from complexing proteins, compared
with another botulinum toxin type A in the treatment of
glabellar frown lines. Dermatologic Surgery 36:2146–2154
Stengel G, Bee K, 2011 Antibody-induced secondary treatment
failure in a patient treated with botulinum toxin type A for
glabellar frown lines. Journal of Clinical Interventions in Aging
6:281–284
Wohlfarth K, Muller C, Sassin I, et al 2007 Neurophysiological
double-blind trial of a botulinum neurotoxin type a free of
complexing proteins. Clinical Neuropharmacology 30:86–94
(NT 201) compared with commercially available botulinum
toxin type A (BOTOX) in healthy volunteers. Journal of Neural
Transmission 112:905–913
Kanovsky P, Slawek J, Denes Z, et al 2009 Efficacy and Safety of
Botulinum Neurotoxin NT 201 in Poststroke Upper Limb
Spasticity. Clinical Neuropharmacology 35:259–265
Kerscher M, Roll S, Becker A, et al 2012 Comparison of the spread
of three botulinum toxin type A preparations. Archives of
Dermatological Research 304(2):155–161
Lee S-K 2007 Antibody-induced failure of botulinum toxin type A
therapy in a patient with masseteric hypertrophy. Dermatologic
Surgery 33:S105–S110
Prager W, Wissmüller E, Kollhorst B, et al 2011 Treatment of
crow’s feet with two different botulinum toxin type A
preparations in split-face technique. Hautarzt 62:375–379
Prager W, Wissmüller E, Kollhorst B, et al 2010 Comparison of two
botulinum toxin type A preparations for treating crow’s feet:

treat facial rhytides caused by hyperkinetic muscles and
for the management of hyperhidrosis.
Myobloc® is contributed as a purified solution packaged
in ready-to-use vials and requires no reconstitution. The
solution is stabilized at pH 5.6 to ensure that the toxin
complex is intact with a single-entity molecular weight of
approximately 700 kDa. The liquid preparation form
enables the toxin to be injected as a uniform and intact
complex, as opposed to partially dissociated subunits. The
non-lyophilized and non-vacuum-dried preparation also
minimizes the amount of denatured protein and secures
an active neurotoxin amount of 65%. Myobloc® is available
in vials of 2500, 5000, and 10 000 U, each with a concen­
tration of 5000 U/ml. As the toxic potency depends on
several factors and cannot be transferred directly from
animals to humans, the equivalency to BoNT-A-based
Botox® / Xeomin® units is considered to be in a range from
1 : 20 to more than 1 : 125. Myobloc® can be diluted with
saline, as much as sixfold, before injection without loss in
total activity and is stable for 9 months when stored at
room temperature (25°C) and for >36 months when
stored at refrigerated temperatures (2–8°C).
Pharmacology of botulinum toxin B
Botulinum neurotoxins are synthesized as macromolecu­
lar complexes of 300–900 kDa and different neurotoxin
complex protein structures, depending on the serotype of
the clostridial strain that produced it. Every progenitor
toxin contains a 150 kDa neurotoxin molecule and one
or more non-toxin proteins. At approximately 700 kDa
the molecular weight of the BoNT-B complex is in
between that of BoNT-A (900 kDa) and all other sero­
types, with weights <500 kDa. The neurotoxin complex
is composed of associated proteins such as hemagglutinin
and a non-toxic, non-hemagglutinin moiety. The active
neurotoxin exists in a 150 kDa dichain molecule consist­
ing of a heavy chain of approximately 100 kDA and a
light chain of approximately 50 kDA, linked together by
a disulfide bond.
Botulinum toxin B affects the nervous system in a
similar way to all other botulinum toxins in a series of
cellular actions including binding, internalization, and inhi­
bition of acetylcholine release. The heavy chain of the
complex contributes to the irreversible binding of the
toxin to the serotype-specific protein within the soluble
7 
Basic science: Myobloc®
Neil S. Sadick, Nils Krueger
3D basic science: Myobloc®
Myobloc® (Solstice Neurosciences, San Francisco, CA), or
Neurobloc® in Europe (Eisai Europe, Hatfield, UK), is the
only commercially available drug based on botulinum
toxin serotype B (BoNT-B) and was FDA cleared for the
treatment of abnormal head position and related pain of
cervical dystonia in 2000. Besides this indication, it has
been shown to be effective for the treatment of many
other diseases like subacromial bursitis, Frey syndrome,
spastic trismus, Parkinson disease-related drooling or sial­
orrhea in children with cerebral palsy. Similar to drugs
based on botulinum toxin serotype A (BoNT-A), Myobloc®
has also been used in the field of aesthetic dermatology to
Summary and Key Features
•
Myobloc® is the only botulinum toxin product that
is based on serotype B
•
Botulinum toxin B (BoNT-B) has its own unique
mechanism of action, binds specific serotype
receptors, and targets specific intracellular proteins
•
Myobloc® requires no reconstitution before
injecting as it is available in a liquid formulation
•
Myobloc® is FDA approved for the treatment of
abnormal head position and neck pain that
accompanies cervical dystonia in adults
•
BoNT-B is an important replacement whenever it
comes to immunoresistance in patients treated
with BoNT-A
•
Myobloc® has a higher rate of adverse effects and
the injection can be more painful compared with
other BoNT products
•
BoNT-B has a rapid onset of effect (within 48
hours after injection). The duration is limited to a
maximum of 10–12 weeks
•
BoNT-B affects a larger area around the injection
point associated with a smoother clinical effect
•
The most common side effects when used for the
treatment of facial rhytides are xerostomia and
headaches as well as dry eyes and brow ptosis

44
Botulinum toxin
N-ethyl maleimide-sensitive factor attachment protein
receptor (SNARE) complex, which is involved in release
of acetylcholine-containing vesicles from the presynaptic
neuron. The light chain mediates proteolysis of SNARE
proteins and subsequent inhibition of synaptic vesicle
fusion to the presynaptic membrane of the human motor
neurons. This leads to flaccid paralysis associated with
botulism. Each serotype cleaves a specific residue on one
of the N-ethylmaleimide sensitive factor (NSF) attach­
ment proteins. Botulinum toxin B cleaves the vesicle-
associated membrane protein (VAMP) also known as
synaptobrevin. VAMP is cleaved by the serotypes F, G,
and D too, but at differing locations (Table 7.1). The high
specificity of the BoNT light chain cleavage is attributed
to the light chain recognizing long SNARE sequences with
30–50 residues indicated by the specific serotype.
Immunogenicity
Patients treated with any botulinum toxin can develop
neutralizing antibodies that reduce or completely eradi­
cate the toxin’s clinical effectiveness. All botulinum sero­
types are antigenically distinct and can cause an individual
immune response. The formation of antibodies has been
linked to high loads of neurotoxin complex protein. Con­
sequently, botulinum toxin preparations with high specific
potencies and low total unit doses have the lowest immu­
nogenicity. The incidence of antibody formation with
BoNT-A in cervical dystonia was reported by Berman to
be less than 5%. In a study by Jankovic and colleagues, the
immunoresistance rate for BoNT-B was found to be up to
30% after 42 months when cervical dystonia patients were
treated with high doses of Myobloc®. Currently there are
no published data on immunoresistance in patients treated
for aesthetic purposes, but the incidence is considered to
be significantly lower as much lower doses are used and
the development of antibodies correlates with the total
cumulative dose. Patients becoming resistant to one par­
ticular serotype can usually be treated with another sero­
type. As botulinum toxin serotypes share some sequence
homology with one another, a theoretical basis for serum
cross-reactivity is given, which can further potentiate
resistance to other serotypes with multiple injections.
Consequential risk factors associated with the evolution
of cross-resistant antibodies include treatment of high
doses of toxin, increased frequency of administration, and
the amount of neurotoxin protein delivered in each
injection.
Table 7.1  Pharmacology of botulinum toxin serotypes
by target / cleavage sizes
Serotype
Target size
Cleaves at
A
SNAP-25*
Gln197–Arg198
B
VAMP†
Glu76–Phe77
C
Syntaxin
Lys253–Ala254
Lys252–Ala252
SNAP-25*
Arg198–Ala199
D
VAMP†
Ala67–Asp68
Lys59–Leu60
E
SNAP-25*
Arg180–Ile181
F
VAMP†
Gln58–Lys59
G
VAMP†
Ala81–Ala82
*SNAP-25 is a soluble NSF attachment protein of 25 000 kDa.
†VAMP = vesicle-associated membrane protein (synaptobrevin).
Pearl 1 
Resistance to BoNT-A appears more often in the treatment
of movement disorders, but can also appear after aesthetic
treatments. Myobloc® may be helpful in people showing
resistance to botulinum toxin phenotype A.
CASE STUDY 1 
Resistance to BoNT-A
A 38-year-old female presented in the office with a history
of treatments with 25 U onabotulinumtoxinA and 75 U
abobotulinumtoxinA for upper face rejuvenation. She was
unsatisfied because the last treatments had effects for less
than 6 weeks.
Due to the apparent BoNT-A resistance, she was switched
to BoNT-B. After injection of 2500 U BoNT-B in the upper
third of the face, the average duration of effect was 12 weeks.
Myobloc® in the aesthetics practice
Despite its primary indication in the treatment of cervical
dystonia, increasing cosmetic demands for the effective
treatment of facial rhytids have provided Myobloc® with
a platform to expand into the dimension of off-label usage.
BoNT-B has been successfully investigated for the treat­
ment of hyperkinetic rhytides as well as for the therapy
of palmar and axillar hyperhidrosis.
Efficacy in the treatment of
hyperkinetic rhytides
BoNT-B is shown to be efficient in the treatment of hyper­
kinetic rhytides in the upper third of the face. Studies
confirm the denervating effect on orbicularis oculi, fron­
talis, and corrugator supercilii (Table 7.2). The described
efficacy with 2500–3000 U per treatment side is 42–
100% after 24–72 hours, 62–78% after 4 weeks, 26–50%
after 8 weeks and <5% after 12 weeks. Rhytides in the
lower two-thirds of the face have not yet been assessed.

45
7 
Basic science: Myobloc®
Table 7.2  Systematic overview of studies evaluating the effect of BoNT-B on hyperkinetic wrinkles
Author
Year
Objective
Area
Design
Material and
methods
Results
Side effects
Level of
evidence
Choi et al
2011
Effectiveness and
safety of a mixture
of BoNT-A and
BoNT-B
Forehead
rhytides
Double-blind
RCT
n n = 14
n 500 U BoNT-B
+ 5 U BoNT-A
n 10 U BoNT-A
n No side-to-side difference at all
FU (p > 0.05)
n 21% eyelid swelling
(both sides)
n 7% heaviness in
forehead (only BoNT-A)
2b
Oh et al
2011
Effect of BoNT-B
on facial flushing
Facial
erythema
Double-blind
RCT
n n = 15
n 626 U BoNT-B
n Saline as
placebo
n No significant decrease in
erythema
n No difference in pain
n No side effects
2b
Carruthers
et al
2007
Dose finding, safety
and tolerability of
BoNT-B
Glabellar
rhytides
Double-blind
RCT
n n = 139
(BoNT-B or
placebo)
n Total of 250,
500, 1000,
1500, 2000,
2500 or 3000 U
BoNT-B
n Baseline vs week 1, 4, 8
(p = 0.026)
n No sign difference between
250/500 U and placebo at week 1
n Significant increasing dose–
response trend
n 0% after 3 months
n No sign difference in pain to
placebo
n 14% headache (placebo
11%)
n 4% flu-like symptoms
(0%)
n 3% upper respiratory
tract infection (7%)
2b
Alster
et al
2003
Safety and efficacy
of BoNT-B
Glabellar
rhytides
Uncontrolled
n n = 20
n total of 2500 U
BoNT-B
n 42% after 48–72 h
n 78% after 1 month
n 50% after 2 months
n 0% after 4 months
n Moderate injectional
pain
n Rare bruising
n Frontal brow tightness
4
Bauman
et al
2003
Safety and efficacy
of BoNT-B
Crow’s
feet
Double-blind
RCT
n n = 15
(BoNT-B)
n n = 5 (placebo)
n total of 3000 U
BoNT-B
n BoNT-B vs placebo (p < 0.001)
n 62% after 1 month
n 26% after 2 months
n 3% after 3 months
n 0% after 4 months
n 45% dry mouth
n 5% dry eyes
n 25% bruising
n 25% headache
n 2.5% indigestion
n (55% flu-like symptoms)
2b
Flynn et al
2003
Onset and diffusion
of BoNT-A and B
Forehead
rhytides
Double-blind
RCT
n n = 8
n 5 U BoNT-A
n 500 U BoNT-B
n Slightly quicker onset with
BoNT-B
n Greater area of diffusion with
BoNT-B
n na
2b
Kim et al
2003
Dose finding,
efficacy and safety
of BoNT-B
Upper
third of
face
Uncontrolled
n 400, 800, 1600,
2000, 2500 or
3000 U BoNT-B
per side
n Near-complete paralysis in
24–72 h
n Clinically effective for up to
8 weeks with 400 U
n Dose-dependent effect after 8
and 12 weeks with 800 or 1600 U
n No longer duration with >1600 U
n 15% headache
n 13% dry mouth
n 7% heavy brow
n 5% dry eyes
4
Continued

46
Botulinum toxin
Author
Year
Objective
Area
Design
Material and
methods
Results
Side effects
Level of
evidence
Matarasso
2003
Efficacy, onset,
duration and safety
of BoNT-A and
BoNT-B
Crow’s
feet
Double-
blind,
controlled
n n = 10
n 15 U BoNT-A
n 750 U BoNT-B
n 100% higher degree of
discomfort with BoNT-B
n Faster onset with BoNT-B
n Longer duration with BoNT-A
than BoNT-B
n No side effects
2b
Sadick
NS
2003
Dose response,
safety, efficacy and
duration of BoNT-B
Glabellar
rhytides
Uncontrolled
n n = 39
n 2400 or 3000 U
BoNT-B
n Full response at day 2–3
n 9.6 weeks’ duration with 2400 U
BoNT-B
n 10.1 weeks’ duration with 3000 U
BoNT-B
n 7% mild pain (3/39)
n 5% ptosis (2/39)
n 3% headaches (1/39)
4
Lowe et al
2002
Efficacy and
duration of BoNT-A
and BoNT-B
Glabellar
rhytides
Controlled
n n = 9
n 20 U BoNT-A
n 1000 or 2000 U
BoNT-B
n Faster onset with BoNT-B
n Longer duration with BoNT-A
than BoNT-B
n Longer duration with 2000 U
BoNT-B than 1000 U
n 11% (1/9) headaches
(1000 U BoNT-B)
2b
Ramirez
et al
2002
Safety and efficacy
of BoNT-B
Glabellar
rhytides,
forehead
rhytides
or crow’s
fee
Uncontrolled
n n = 24
n 400–800 U
BoNT-B
n Near-complete paresis within
24–72 h
n Duration of effect of 8 weeks
n No side effects
4
Sadick
2002
Safety, onset and
duration of BoNT-B
Glabellar
rhytides
Uncontrolled
n n = 30
n 1800 U BoNT-B
n Onset at day 1–3
n Duration 6–10 weeks
n 7% mild pain (2/30)
n 3% headaches (1/30)
4
Spencer
et al
2002
Safety and
dose–response of
BoNT-B
Glabellar
rhytides
or
forehead
rhytides
Uncontrolled
n n = 42
n 1875, 2500 or
3125 U
(glabellar)
n 2250, 3000 or
3750 U
(frontalis)
n Duration of effect between 8 and
12 weeks
n Longer duration with higher dose
n No correlation between dose and
pain
n No ptosis or dropped
brow
4
Table 7.2  Systematic overview of studies evaluating the effect of BoNT-B on hyperkinetic wrinkles —cont’d

47
7 
Basic science: Myobloc®
Referring to the study by Carruthers et al, a dose of at
least 1500 U BoNT-B seems to be ideal for a complete
paresis of the frontalis muscle. Direct comparisons with
BoNT-A in a 1 : 50 ratio show similar efficacy in the first
4 weeks after injections, but an inferiority of BoNT-B at
later timepoints.
Dose response
All studies assessing the dose response show a clear
increase of duration with higher doses. While Kim et al
approved increasing duration with doses from 400 to
1600 U BoNT-B per treated side, Spencer et al found this
correlation for higher doses from 1875 to 3750 U to be
effective, too. Carruthers et al confirmed these findings in
one study with 139 subjects for six different doses from
500 to 3000 U, but did not find a significant difference
between 250 and 500 U.
Onset and duration
Onset and duration of effect of BoNT-B have been assessed
in several studies. The agent is described as taking effect
rapidly after injection. Near-complete paresis of the
treated muscle is usually seen within 24–72 hours after
injection. In direct comparison to BoNT-A, the onset of
BoNT-B is quicker and the clinical result is smoother. The
duration of effect depends on the dosage used, but seems
to be limited to a maximum of 10–12 weeks. Sadick (in
2003) specified the duration of effect with 2400 U
BoNT-B to be 9.6 weeks, while it was 10.1 weeks with
3000 U. Bauman et al found an average wear-off time of
7 weeks after injection of 1500 U BoNT-B into each orbi­
cularis oculi. While studies by Comella et al and Tintner
et al show a superior duration of BoNT-A in patients
treated for cervical dystonia, a study by Pappert et al
shows no difference. The non-inferiority is supported by
an animal study by Arezzo, which shows a consistent dura­
tion of the direct effects on the treated muscle when
equivalent doses of BoNT-A or BoNT-B were injected.
Diffusion and spreading
The effect of botulinum toxin is not limited to the injec­
tion site. As the muscle fascia is a relatively ineffective
boundary the unbound toxin can, driven by the concentra­
tion gradient and the dynamics of the injection, diffuse
to nearby cholinergic neurons through the extracellular
space. The spreading is not limited to tissue in close prox­
imity, as distant muscles can be affected due to vascular
or lymphatic transport of the toxin. Because of the
smoother and less precise clinical effect, BoNT-B is
assumed to diffuse more widely than does BoNT-A. This
theory is supported by the high rate of dry mouth or dry
eyes after injections with BoNT-B, reported in studies by
Baumann et al and Kim et al, as well as the finding by
Chapman et al of dysphagia as a common adverse effect
in the treatment of cervical dystonia with BoNT-B. Doubts
on the higher diffusion potential come from Arezzo’s
animal studies, as his studies show that BoNT-A can cause
a paralysis up to 40% in non-treated, contralateral muscles
compared with 0% with BoNT-B at equivalent doses.
These data indicate that differences in the pattern of side
effects for BoNT-A and BoNT-B may be more related to
differences in receptor distribution on autonomic neurons
than to greater spread of paralysis from an injection site.
Pearl 2 
BoNT-B is known to be the botulinum toxin phenotype with
the fastest onset time. It usually takes effect in the first
48 hours after injection. Myobloc® may therefore be helpful
for people who want a more rapid onset.
CASE STUDY 2 
Fast Onset
A 42-year-old female presented in the office for forehead and
glabella rejuvenation, in preparation for her daughter’s
wedding 2 days later. Because the onset of BoNT-A is
delayed, she was treated instead with a total of 3125 U
BoNT-B for the interglabellar complex and 2500 U BoNT-B
for the frontalis BoNT-B. The treatment effect was apparent
in time for the ceremony.
Pearl 3 
BoNT-B is known to be effective in a larger area around the
injection site. The effect is smoother and more natural.
Myobloc® should therefore be used where a greater spread
and more natural appearance is wanted.
Tolerability and safety
Injections with BoNT-B are mainly reported to be more
painful than injections with other botulinum toxins. It is
understood that the increased pain is not related to the
toxin itself, but rather to the acidic pH of 5.6. Overall,
the injection pain is described to be mild to moderate and
for most patients no reason to discontinue the therapy.
However, a double-blind study by Carruthers et al showed
no difference in pain between BoNT-B and placebo.
The safety of botulinum toxin B is well examined. The
most common side effects, when used in aesthetic medi­
cine, are dry mouth (13–45%), headaches (3–25%), dry
eyes (5%), as well as heavy brows and lid ptosis (5–7%).
Furthermore, two studies (by Baumann et al and
Carruthers et al) describe flu-like symptoms after the
injection of BoNT-B, but relate it to the seasonal context.
The described side effects are comparable to those seen
in the treatment of chronic neurological movement dis­
orders, where doses of up to 25 000 U BoNT-B are not
uncommon.
Pearl 4 
Due to the larger area of effect of BoNT-B, Myobloc® should
be used with caution in elderly people where the risk of brow
ptosis is higher.

48
Botulinum toxin
Myobloc® in the treatment of hyperhidrosis
Beside its use for the treatment of hyperkinetic facial
lines, BoNT-B is effective for the treatment of hyperhi­
drosis. The eccrine sweat glands are innervated by sympa­
thetic nerves that use acetylcholine. As botulinum toxin
has the ability to block the release of this neurotransmitter
in the postganglionic sympathetic fibers of the glands, it
can be used to stop the excessive sweat production for
several weeks (Table 7.3).
For the treatment of focal palmar or axillary hyperhi­
drosis, injected botulinum toxin has to be evenly distrib­
uted every 2 cm2 in the areas in which a starch iodine test
indicated sweating. A sweat reduction of 95–100% with
a mean duration of 3–5 months can be expected when
doses of 2000–5000 U BoNT-B are used. Hechtel et al
showed an extremely low-dose treatment with only 250 U
BoNT-B per axilla to be effective for 1–3 months. However,
there seems to be no correlation between dosage and
duration or efficacy, when higher doses (2000 U per side)
are used.
Pearl 5 
The larger area of effect of BoNT-B is particularly useful in
the treatment of hyperhidrosis as fewer injections per
treatment area are needed.
The information on safety is highly variable. The most
commonly described adverse effect is xerostomia, occur­
ring in 5–90% of subjects. Other common side effects
are heartburn, excessively dry hands, muscle weakness,
decreased grip strength, dry eyes, acne, headache, and
flu-like symptoms. However, there are also studies (by
Hecht et al and Frasson et al) where no side effects other
than mild pain at the injection side occurred. In direct
comparison with BoNT-A, BoNT-B seems to have a faster
onset and a shorter duration of effect.
Treatment considerations for Myobloc®
A good rule of thumb to produce Myobloc® results com­
parable to BoNT-A is to use the established total of
Botox® / Xeomin® units and multiply it by 100 to find the
number of Myobloc® units needed. As the clinical effect
of Myobloc® is wider than that of BoNT-A, the number of
injection points can be reduced in most cases (Table 7.4).
According to this rule, to eradicate glabellar lines the pro­
cerus complex and corrugators supercilii muscles have to
be treated with 2000–3000 U Myobloc® in three or four
injection sites instead of 20–30 U Botox® / Xeomin® units
in five injection sites. The injection of the frontalis utilizes
1500–3000 U of Myobloc®, distributed evenly over three
or four injection sites to provide satisfactory results. A
dose of 1500 U of Myobloc® has to be injected on each
side of the face, into the corrugator supercilii, the procerus
complex, and the medial portion of the orbicularis oculi
muscle, to achieve effective results in the lifting of the
brow. It is found that, for the treatment of crow’s feet,
1000–2000 U of Myobloc® per side have to be injected
into two to three sites of each orbicularis oculi muscle.
Summary
Myobloc® is the only commercial available drug based on
botulinum toxin serotype B. It is FDA cleared for the treat­
ment of cervical dystonia, but also applicable for the treat­
ment of hyperkinetic facial rhytides and focal hyperhidrosis
of the palms and axillae. In contrast to other botulinum
toxin products, Myobloc® is available as a purified solution
packaged in ready-to-use vials of 2500, 5000, and 10 000 U
each, requiring no reconstitution. The liquid preparation
ensures the integrity of the toxin as uniform and intact
complex and minimizes the amount of denatured protein.
The equivalency of Myobloc® units to the widely used
Botox® / Xeomin® units is reported to be in a range between
1 : 20 and 1 : 125 and mostly said to be 1 : 100.
Myobloc® is shown to be efficient in the treatment
of hyperkinetic rhytides of the upper third of the face
in several clinical studies. It is described to have a faster
onset and a smoother and less narrow effect, but also a
shorter duration of effect, when compared with BoNT-A.
However, the duration increases with higher doses, but
seems to be limited to a maximum of 10–12 weeks. The
diffusion potential of Myobloc® is unclear. Due to the
wider effect and the higher rate of side effects, BoNT-B
is assumed to exhibit more diffusion and spread. However,
these findings can also be related to differences in receptor
distribution on autonomic neurons.
The safety profile of Myobloc® is well examined. The
most common side effects when used for the treatment
of facial rhytides are xerostomia and headaches as well as
dry eyes and brow ptosis. Injections with Myobloc® are
mainly reported to be more painful than injections with
other botulinum toxins. Patients describe the injection
pain as mild to moderate. It is assumed that the increased
pain is related not to the toxin itself, but to the solution’s
acidic pH of 5.6.
In addition to Myobloc’s application for muscle dener­
vation, it is also effective for the treatment of focal hyper­
hidrosis, due to its ability to block the sweat glands’
neurotransmitter, acetylcholine. A near-complete sweat
reduction with a duration of 3–5 months can be expected,
when doses of 2000 Myobloc® units per palm or axilla are
injected. In direct comparison with products based on
BoNT-A, Myobloc® is reported to have a faster onset and
a longer duration of effect. The safety profile for the treat­
ment of hyperhidrosis is generally equivalent to that for
the treatment of facial rhytides. Additionally, reported
effects are excessively dry hands, muscle weakness, and
decreased grip strength.
Conclusion
In times where the average life expectancy is increasing
greater than ever in history, a young and beautiful

49
7 
Basic science: Myobloc®
Table 7.3  Systematic overview of studies evaluating the effect of BoNT-B on hyperhidrosis
Author
Year
Objective
Area
Design
Material and
methods
Results
Side effects
Level of
evidence
Frasson
et al
2011
Efficacy and
duration of
BoNT-A and -B
Axilla
Single-blind
RCT
n n = 10
n 50 U BoNT-A vs
2500 U BoNT-B
n Higher sweat reduction with
BoNT-B
n Higher patient satisfaction
with BoNT-B
n Faster onset and longer
duration with BoNT-B
n Mild pain, especially with BoNT-B
n No systemic adverse effects
2b
Bauman
et al
2005
Safety, efficacy
and duration of
BoNT-B
Palm
Double-blind
RCT
n n = 20
n 5000 U BoNT-B per
palm or placebo
n Significant sweat reduction in
30 days
n Mean duration of 3.8 months
n 90% dry mouth
n 60% heart burn
n 60% excessively dry hands
n 60% muscle weakness
n 50% decreased grip strength
n 35% dry eyes
n 15% acne
n 5% flu-like symptoms
2b
Nelson
et al
2005
Safety and
efficacy of
BoNT-A and -B
Axilla
Uncontrolled
n n = 13
n 5000 U BoNT-B
n Significant sweat reduction
n >80% reduction after 12
weeks
n 85% dry mouth (11/13)
n 15% headache (2/13)
n 15% increased sweating on
untreated axilla (2/13)
n 8% sensory-motor symptoms of
hand (1/13)
4
Bauman
et al
2005
Safety, efficacy
and duration of
BoNT-B
Axilla
Uncontrolled
n n = 20
n 2500 U BoNT-B per
axilla or placebo
n Significant sweat reduction in
30 days
n Mean duration of 5 months
n 35% dry mouth
n 30% flu-like symptoms
n 25% dry eyes
n 25% bruising
n 20% headache
n 20% acne
n 15% indigestions
4
Hecht
et al
2004
Efficacy and
duration of
BoNT-B
Axilla
Uncontrolled
n n = 4
n 250 U BoNT-B per
axilla
n 95–100% sweat reduction
after 21 days
n Duration of 1–3 months
n No side effects
4
Dressler
et al
2002
Safety, efficacy,
onset and
duration of
BoNT-A and B
Axilla
CT
n n = 19
n 100 U BoNT-A vs
4000 U BoNT-B
n 100 U BoNT-A vs
2000 U BoNT-B
n No difference between toxins
in effect or duration
n Earlier onset with BoNT-B
n Mean duration of 4 months
n More discomfort with BoNT-B
n 5% dry mouth (1/19)
n 5% accommodation difficulties
(1/19)
n 5% conjunctival irritation (1/19)
2b

50
Botulinum toxin
appearance is not only associated with fertility and good
health, but can also go with social and economic advan­
tages. Recognition of this fact has created a strong public
demand for safe and effective aesthetic solutions for the
treatment of wrinkles. One of them is the treatment of
overactive facial muscles that cause hyperkinetic rhytides
with botulinum toxin to decrease the muscles’ activity and
cause paralysis.
Among the botulinum toxins available in the United
States, Myobloc® is the only product that is based on
botulinum toxin serotype B. It has its own unique mecha­
nism of action, binds specific serotype receptors, and
targets specific intracellular proteins. It is also the only
botulinum toxin product that is available in a liquid for­
mulation and therefore requires no reconstitution before
injecting.
Its unique characteristics and clinical profile, including
its very fast onset, smooth result, and superiority in the
treatment of hyperhidrosis, make Myobloc® an interesting
alternative to BoNT-A-based products. Furthermore, it
is an important replacement whenever there is immuno­
resistance found to be in patients treated with BoNT-A.
However, it should be remembered that Myobloc® has a
higher rate of adverse effects and the injection can be
more painful compared with other BoNT products.
Although the likelihood of serious adverse events is
low, all botulinum toxins are considered to be highly
potent neurotoxins and extreme care should be taken,
especially in increasing doses in patients with any indica­
tions including off-label usage. Of course, as physicians
become more vigorous with toxin usage for aesthetic
treatments (especially in the lower face), the side effects
become prevalent and troublesome. For a physician in the
aesthetic arena, not only is patient satisfaction important
but also patient safety is imperative – especially a thor­
ough understanding of practice and procedure and so
avoidance of post-treatment complications.
Further reading
Alster TS, Lupton JR 2003 Botulinum toxin type B for dynamic
glabellar rhytides refractory to botulinum toxin type A.
Dermatologic Surgery 29(5):516–518
Table 7.4  Provisional dosing guidelines for botulinum
toxin B injections for facial rhytids
Muscle site
BoNT-B (Myobloc®)
Units
No. of injections
Glabella
2000–3000
3
Frontalis
1500–3000
3–4
Brow lift
1500 total
2–4 per side
Periorbital
1000–2000 per side
2–3 per side
Aoki KR, Guyer B 2001 Botulinum toxin type A and other
botulinum toxin serotypes: a comparative review of biochemical
and pharmacological actions. European Journal of Neurology
8(suppl 5):21–29
Arezzo JC 2009 NeuroBloc / Myobloc: unique features and findings.
Toxicon 54(5):690–696
Baumann L, Slezinger A, Halem M, et al 2005 Pilot study of the
safety and efficacy of Myobloc (botulinum toxin type B) for
treatment of axillary hyperhidrosis. International Journal of
Dermatology 44(5):418–424
Baumann L, Slezinger A, Halem M, et al 2005 Double-blind,
randomized, placebo-controlled pilot study of the safety and
efficacy of Myobloc (botulinum toxin type B) for the treatment
of palmar hyperhidrosis. Dermatologic Surgery 31(3):263–270
Baumann L, Slezinger A, Vujevich J, et al 2003 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. Dermatologic Surgery
29(5):508–515
Berman B, Seeberger L, Kumar R 2005 Long-term safety, efficacy,
dosing, and development of resistance with botulinum toxin
type B in cervical dystonia. Movement Disorders 20(2):233–
237
Callaway JE 2004 Botulinum toxin type B (Myobloc):
pharmacology and biochemistry. Clinical Dermatology
22(1):23–28
Carruthers A, Carruthers J, Flynn TC, et al 2007 Dose-finding,
safety, and tolerability study of botulinum toxin type B for the
treatment of hyperfunctional glabellar lines. Dermatologic
Surgery 33(1 spec no.):S60–S68
Chapman MA, Barron R, Tanis DC, et al 2007 Comparison of
botulinum neurotoxin preparations for the treatment of cervical
dystonia. Clinical Therapeutics 29(7):1325–1337
Choi J-W, Youn C-S, An H-T, et al 2011 Combined use of
botulinum toxin type A and B for forehead rhytides: a
randomized, double-blind, split-face study. Journal of
Dermatological Treatment. Online. Available:
nlm.nih.gov/pubmed/21801115, 20 October 2011 [Epub ahead
of print]
Comella CL, Jankovic J, Shannon KM, et al 2005 Comparison of
botulinum toxin serotypes A and B for the treatment of cervical
dystonia. Neurology 65(9):1423–1429
Dressler D, Adib Saberi F, Benecke R 2002 Botulinum toxin type B
for treatment of axillar hyperhidrosis. Journal of Neurology
249(12):1729–1732
Flynn TC, Clark RE 2nd 2003 Botulinum toxin type B
(MYOBLOC) versus botulinum toxin type A (BOTOX) frontalis
study: rate of onset and radius of diffusion. Dermatologic
Surgery 29(5):519–522; discussion 522
Frasson E, Brigo F, Acler M, et al 2011 Botulinum toxin type A vs
type B for axillary hyperhidrosis in a case series of patients
observed for 6 months. Archives of Dermatology
147(1):122–123
Halpern JL, Smith LA, Seamon KB, et al 1989 Sequence homology
between tetanus and botulinum toxins detected by an
antipeptide antibody. Infection and Immunity 57(1):18–22
Hecht MJ, Birklein F, Winterholler M 2004 Successful treatment of
axillary hyperhidrosis with very low doses of botulinum toxin B:
a pilot study. Archives of Dermatological Research
295(8-9):318–319
Jankovic J, Hunter C, Dolimbek BZ, et al 2006 Clinico-
immunologic aspects of botulinum toxin type B treatment of
cervical dystonia. Neurology 67(12):2233–2235
Kim EJ, Ramirez AL, Reeck JB, et al 2003 The role of botulinum
toxin type B (Myobloc) in the treatment of hyperkinetic facial
lines. Plastic and Reconstructive Surgery 112(5 suppl):88S–93S;
discussion 94S–97S

51
7 
Basic science: Myobloc®
Kranz G, Paul A, Voller B, et al 2011 Long-term efficacy and
respective potencies of botulinum toxin A and B: a randomized,
double-blind study. British Journal of Dermatology
164(1):176–181
Lowe NJ, Yamauchi PS, Lask GP, et al 2002 Botulinum toxins
types A and B for brow furrows: preliminary experiences with
type B toxin dosing. Journal of Cosmetic and Laser Therapy
4(1):15–18
Matarasso SL 2003 Comparison of botulinum toxin types A and
B: a bilateral and double-blind randomized evaluation in the
treatment of canthal rhytides. Dermatologic Surgery 29(1):7–
13; discussion 13
Nelson L, Bachoo P, Holmes J 2005 Botulinum toxin type B: a new
therapy for axillary hyperhidrosis. British Journal of Plastic
Surgery 58(2):228–232
Oh YJ, Lee NY, Suh DH, et al 2011 A split-face study using
botulinum toxin type B to decrease facial erythema index.
Journal of Cosmetic and Laser Therapy 13(5):243–248
Pappert EJ, Germanson T 2008 Botulinum toxin type B vs. type A
in toxin-naïve patients with cervical dystonia: randomized,
double-blind, noninferiority trial. Movement Disorders
23(4):510–517
Ramirez AL, Reeck J, Maas CS 2002 Botulinum toxin type B
(MyoBloc) in the management of hyperkinetic facial
lines. Otolaryngology Head and Neck Surgery 126(5):459–
467
Sadick NS 2003 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. Dermatologic Surgery
29(5):501–507; discussion 507
Sadick NS 2002 Botulinum toxin type B for glabellar wrinkles: a
prospective open-label response study. Dermatologic Surgery
28(9):817–821
Spencer JM, Gordon M, Goldberg DJ 2002 Botulinum B treatment
of the glabellar and frontalis regions: a dose response analysis.
Journal of Cosmetic and Laser Therapy 4(1):19–23
Tintner R, Gross R, Winzer UF, et al 2005 Autonomic function
after botulinum toxin type A or B: a double-blind, randomized
trial. Neurology 65(5):765–767

Clostridium botulinum to the Korean Advanced Institute
of Science and Technology (KAIST) with him after several
years of research at the University of Wisconsin. While
the C. botulinum produced at the University of Wisconsin
became the source of Oculinum® – the first licensed botu­
linum toxin product, later renamed Botox®, the exact
same strain at KAIST became the foundation of botulinum
toxin research in Korea as well as the source of Neuro­
nox®, which was formulated by a group of scientists from
KAIST, who later founded Medytox Inc. As a result the
C. botulinum type A Hall strain used by Medytox Inc.
(GenBank accession number DQ409059) is exactly the
same bacterial strain as that used by Allergan (AF488749,
488748, 488747, AF488746, AF488745), and there is no
difference between the two deduced amino acid sequences
derived from the DNA sequence of the type A toxin
complex.
However, differences in manufacturing processes, such
as purification and filtration, can result in differences in
the bulk toxin even amongst those from the same culture
solution. One important physicochemical feature poten­
tially affected by such processes would be the molecular
weight. The size exclusion high-performance liquid chro­
matography (SE-HPLC) analysis of Neuronox® yields a
single peak in the chromatogram with an average molecu­
lar weight of 904 ± 7 kDa. This is highly comparable with
that of Allergan: 880kDa by SE-HPLC analysis and 925
± 45 kDa by light-scattering analysis.
In addition, the botulinum toxin is formulated in dif­
ferent ways in the final stages. Neuronox® is formulated
using a freeze-drying method, whereas Botox® is formu­
lated as vacuum-dried powder. Nevertheless, Neuronox®
ultimately has a similar formulation to Botox®, with
one vial containing 100 mouse units of the purified toxin
complex, 0.9 mg of sodium chloride, and 0.5 mg of
human serum albumin.
It is still unclear whether the origin of the bacterial
strain, molecular weight, or product formulation is
truly clinically significant. But such specifications sup­
ported by clinical data could provide outline guidelines to
practicing physicians.
Clinical studies for Neuronox®
The safety and efficacy of Neuronox® were compared
with Botox® in two randomized, double-blind studies, at
8 
Medytoxin / Neuronox®
Kyle Koo-II Seo, Wooshun Lee
Introduction
Neuronox® (Medytox Inc., Ochang, South Korea) is a
botulinum toxin type A (BoNT-A) product first approved
in 2006 by the Korean Food and Drug Administration.
Since then, it has been approved under different brand
names as Botulift®, Siax®, Cunox®, and Meditoxin® in 23
countries world wide (Fig. 8.1). Among currently available
BoNT-A products, Neuronox® seems to be one of the
viable alternatives to Botox® in terms of microbiological,
physicochemical, and biochemical features, and clinical
interchangeability in 1 : 1 dose ratio.
Microbiological, physicochemical, and
biochemical profiles
Considering that botulinum toxin is a biological agent
produced in living cells, one ought to first analyze the
bacterial strain that produced the toxin itself. In the
late 1970s, Dr Kyu-Hwan Yang, a microbiologist brought
Summary and Key Features
•
Neuronox® is a botulinum toxin type A (BoNT-A)
product approved in 23 countries
•
Neuronox® shows similar microbiological,
physicochemical, and biochemical features to
Botox®
•
Neuronox® has proven its non-inferiority to Botox®
in terms of efficacy and safety in various clinical
studies in 1 : 1 dose ratio
•
The recommended dosage of botulinum toxin type
A for wrinkle treatment in Asians is usually lower
than that in Caucasians
•
The combination technique of multiple intradermal
injection of botulinum toxin type A with conventional
intramuscular injection is widely utilized in Asia for
the purpose of facial rejuvenation
•
The use of botulinum toxin type A to reduce the
volume of muscles such as masseter, temporalis,
calf, and deltoid is becoming more popular and
promise to have increasingly broader applications

53
8 
Medytoxin / Neuronox®
satisfaction for up to 16 weeks. Both products were well
tolerated (publication in progress). Interestingly, the
response rates at maximum frown at 4 weeks were higher
in this study (93.8% in the Neuronox® group and 94.6%
in the Botox® group) than those conducted in the USA
(76.7% and 83.7% with Botox®) and comparable with
those from China and Japan (88.6%, 95.1%, and 94.1%
with Botox®). All these studies had the same timepoint,
method of assessment, and the definition of ‘responder’,
but different demographics (Asian versus Caucasian).
Based on these clinical data, we could conclude that
the efficacy and safety of Neuronox® are comparable
with those of Botox® not only in the treatment of blepha­
rospasm and muscle spasticity in cerebral palsy, but
also in the treatment of wrinkles such as glabellar lines
and crow’s feet.
Reconstitution and handling
Neuronox® is supplied as a single-use vial containing 50,
100, or 200 units of BoNT-A complex. The package insert
for Neuronox® recommends reconstituting it with non-
preserved 0.9% sodium chloride injection. In a 2009
survey of 500 Korean dermatologists and plastic surgeons,
49.8% of responders dilute a 100 unit vial with 2.5 mL,
yielding 4 units per 0.1mL, and 20.6% of responders
dilute a 100 units vial with 2 mL yielding 5 units per
0.1 mL. Unreconstituted Neuronox® must be stored in a
refrigerator at 2–8°C or in a freezer at below −5°C. Once
reconstituted, the product should be stored at 2–8°C for
later use. The package insert recommends using the recon­
stituted product within 24 hours.
The use of Neuronox® in Asians
Although many clinicians in Asia use Neuronox® and
Botox® interchangeably, their practice does not necessarily
coincide with the published guidelines for Botox®. We
a 1 : 1 dose ratio, for the treatment of essential blepharo­
spasm and spasticity in children with cerebral palsy (Kim
et al and Yoon et al). Neuronox® was proven to be non-
inferior to Botox® in both studies, and there was no
significant difference in the safety profiles. Although pub­
lished studies on Neuronox® are limited to the therapeutic
area owing to its recent introduction to the market, several
clinical studies using Neuronox® in aesthetic treatment
have already been performed and their results are in the
process of publication.
The safety and efficacy of Neuronox® in aesthetic
applications were presented at the 22nd World Congress
of Dermatology in 2011. A total of 49 women with sym­
metric crow’s feet were enrolled in a double-blind, ran­
domized, intraindividual controlled study. Patients’ crow’s
feet were treated with 12 units of Botox® on one side and
with same dose of Neuronox® on the other side. Clinical
improvement was assessed by blinded investigators based
on photographs taken at 1, 4, and 12 weeks post-treatment
on a four point grading scale from poor to excellent; at all
timepoints, there was no significant difference between
two treatments (p > 0.05). At 4 weeks post-treatment,
95.7% of the sides treated with Botox® and 100% of those
treated with Neuronox® demonstrated a good or excellent
result, with 42.6% of either group having an ‘excellent’
result. Also, both products were well tolerated without
any clinically significant treatment-related adverse events.
In another clinical trial, Neuronox® was also directly
compared with Botox® at 1 : 1 dose ratio for the treatment
of glabellar lines in a double-blind, randomized Phase III
study. A total of 314 patients aged 20 to 65 with moderate
to severe glabellar lines were randomly treated with 20
units of Neuronox® or Botox® and assessed by blinded
investigators. Neuronox® was again proven to be non-
inferior to Botox® without any significant difference in any
of the efficacy end-points, including live assessment at
maximum frown and at rest, photographic assessment,
and the subject’s self-assessment of improvement and
Figure 8.1  Neuronox® is marketed under different brand names such as Botulift®, Siax®, Cunox®, and Meditoxin®.

54
Botulinum toxin
retaining a natural expression. This is applied with the
expectation of not only the reduction of dynamic facial
wrinkles but also the reduction of static wrinkles and pore
size, as well as creating the so-called perceived ‘lifted
effect’ – even though the actual lifting effects have not
been objectively verified.
The hypothesized effects of multiple intradermal injec­
tions can be classified into three categories. The first is the
same dynamic wrinkle reduction as delivered by conven­
tional intramuscular injection of BoNT-A. The second is
the improvement of static wrinkles and tightening of
pores, which imparts shine and a tighter look to the skin.
The last is the improvement of facial contours resulting in
a lifted and younger appearance via the elevation of the
lateral part of eyebrow, sharper definition to the chin line
by weakening mentalis and platysma muscle and reducing
the volume of the masseter muscle.
The exact mechanism by which the improvement in
skin texture, including static wrinkles and pores is attained,
has not yet been elucidated. Our hypothesis involves
dermal edema resulting from the transient and mild lym­
phatic insufficiency induced by underlying muscular paral­
ysis and the smooth muscle relaxation of the dermal
vasculature. Dermal edema might improve the fine static
wrinkles and dilated pores. In our recent study on skin
changes post intradermal injections of Neuronox®, its effi­
cacy in static wrinkle depth was demonstrated in a ran­
domized double-blinded, placebo-controlled, split-face
clinical study design (publication in progress). Other
parameters related to skin changes, such as elasticity,
hydration, sebum expression, skin pore size, and skin
thickness, failed to show any significant changes. However,
we note that this study treated only a limited area of the
lateral cheek, and thus is not appropriate for evaluating
sebum production and pore size. Further studies with a
modified regimen are required for capturing those
variables.
Combining multiple intradermal injections with the
conventional intramuscular injection seems more promis­
ing than using either method in isolation. Conventional
intramuscular injections can be used for supplementation
in areas where more targeted paralysis is needed (Fig. 8.2).
This could tailor a more comprehensive approach to using
BoNT for facial rejuvenation.
The third striking feature of BoNT-A treatment in
Asians is its expanding role in facial and body contouring.
Since Dr Smyth et al first reported the use of BoNT-A
to treat masseteric hypertrophy with favorable results,
BoNT-A has proven reduce muscle volume through disuse
atrophy. This novel method has become a great success
among Asians, whose faces tend to be wider or flatter
compared with Caucasians and who aspire to having a
more oval and slimmer face. Before BoNT-A became
widely used for masseter reduction, mandibular angle
resection surgery was the mainstay of treatment. Consid­
ering the downtime, risks, and costs, BoNT-A treatment
for masseteric hypertrophy is far more attractive than
surgery. In patients where muscles contribute more to
identified the following cases with regard to the aesthetic
use of Neuronox® in Asians, which is thought to be more
due to ethnic differences than to a difference between the
products themselves. This might also be valid for other
botulinum toxin products.
First, the optimal dose and injection sites for treating
wrinkles are different from the guidelines advocated in
North America or Europe. For example, when treating
forehead wrinkles, parts of the frontalis lateral to the mid-
pupillary lines are areas not recommended for Caucasians,
but in Asians are routinely treated in conjunction with the
medial parts in order to avoid the so called ‘samurai
eyebrow’, referring to the exaggerated elevation of the
lateral part of the eyebrows following injection of BoNT-A
into the forehead. Because Asians have a wider and flatter
face compared with Caucasians, the ‘samurai eyebrow’
becomes more evident and results in an malevolent
appearance in Asians.
Another concern is the optimal dose of BoNT-A for the
treatment of wrinkles. Although more recently published
recommendations by Carruthers et al in 2004 and 2007
modified the original techniques and decreased dosage of
BoNT-A, the optimal initial doses for wrinkle reduction
in Asians seems to be still lower than that of Caucasians
(Table 8.1). From the above-mentioned data of clinical
studies on the treatment of glabellar wrinkles using
BoNT-A, we found differences in the dosages of BoNT-A
between Asians and Caucasians. Intrinsic ethnic differ­
ences in the muscle mass as well as cultural factors, such
as Asians being less facially expressive, could be the reason
why lower doses are more appropriate.
Secondly, the practice of using multiple intradermal
injections of BoNT-A has been widely adopted in Asia,
under various names such as ‘mesobotox’, ‘dermotoxin’,
or ‘Botox lift’. Asian physicians have incorporated the
technique of multiple intradermal injections of BoNT-A
as an additional solution to achieve subtle changes while
Table 8.1  Recommended dose of BoNT-A for the
reduction of dynamic wrinkles in Asians
Wrinkles
Muscles
Initial dose
(in units)
Forehead wrinkles
Frontalis
3–10
Glabellar frown
lines
Corrugator and procerus
10–14
Crow’s feet
Orbicularis oculi
6–8
Nasal bunny lines
Nasalis
6
Perioral wrinkles
Orbicularis oris
2–4
Cobbled chin
Mentalis
6–8
Neck platysmal
bands
Platysma
20–30

55
8 
Medytoxin / Neuronox®
Figure 8.3  Botulinum toxin type A for
masseteric hypertrophy dramatically
changes lower facial contours: left,
pretreatment and right, 3 months
post-treatment.
Table 8.2  Recommended dosage of BoNT-A for the
reduction of muscle volume.
Muscle
Dose of botulinum toxin type A per side
(in units)
Masseter
10–40
Temporalis
20–40
Gastrocnemius
70–150
Pearl 2 
Sunken cheeks are another potential side effect of treating
masseter hypertrophy with BoNT-A. This can be aesthetically
undesirable because it can impart a gaunt, aged, and
fatigued look. However, this condition is due rather to the
surfacing of a pre-existing condition that was previously
masked by the volume of masseter muscle. Therefore it is
important for physicians to mention such pre-existing
condition to susceptible patients and to inform them of the
need for a subsequent fat graft or filler injection before
treatment with BoNT-A.
Pearl 1 
An unnatural or asymmetric smile after treatment of masseter
hypertrophy with BoNT-A is the most embarrassing side
effect of this treatment; it seems to occur mainly due to the
inadvertent diffusion of BoNT-A into the risorius muscle
overlying the masseter muscle. This can be prevented if the
toxin is injected into the masseter muscle in sufficient depth
by inserting the needle perpendicularly to the skin and
advancing it to touch the mandibular bone. One should
choose the right needle length, at least half an inch (1.25 cm),
for this purpose. Another important tip is to screen out high
risk patients using this simple test: those patients whose ears
move when they smile fully and pull their lips outward more
than others tend to have more developed risorius muscle,
and thus are more at risk. We advise trying with a reduced
dose of less than 10 units per side in those patients.
Figure 8.2  A combination of multiple intradermal and conventional
intramuscular injections of botulinum toxin type A. The green dots
indicate intradermal injection sites (large green dots, 0.4 units; small
green dots, 0.2 units) whereas red dots indicate intramuscular
injection (large red dots, 5–10 units; medium red dots, 3 units; small
red dots, 2 units).
their lower facial contours than bone or fat, BoNT-A treat­
ment can change their appearance dramatically (Fig. 8.3).
Indeed, it is highly suitable for Asians, considering that
even normal Asians have masseteric muscles 0.8 cm thick
per side.
Usually 10–40 units per side are required, depending
on masseteric volume (Table 8.2). In our clinical study
using three-dimensional computed tomography, the overall
masseter muscle volume had decreased by 27.5%
on average at 12 weeks’ post-treatment with 20–40 units
of Neuronox®. Volume reduction of the lower part of
masseter, lying below an imaginary line connecting the
anterior tragus and the corner of the mouth, attributed
more to the contouring of the lower face, by up to 35.1%
(Fig. 8.4) (publication in progress). Other published
studies yielded comparable results. The duration of effect
varied from 3 to more than 12 months depending on the
individual’s dietary habits, bruxism, and so forth. Although
this procedure is generally considered simple and safe,
the possibility of adverse effects should be anticipated
before proceeding with treatment and our suggestions
considered.

56
Botulinum toxin
Figure 8.4  Reduction in masseteric volume
post-injection with Neuronox® demonstrated
on 3-dimensional computed tomography
(upper) and computed tomography scans
of transverse section (lower). The right
masseter muscle was treated with 20 units
and the left masseter muscle was treated
with 30 units of Neuronox®. In both sides a
third of the total muscle volume was
reduced from baseline: left, pretreatment,
and right, 3 months post-treatment.
Figure 8.5  Bulging of medial gastrocnemius
was improved with 100 unit of BoNT-A
injections per side: left, pretreatment; right,
3 months’ post-treatment.

57
8 
Medytoxin / Neuronox®
The rapidly increasing demand for aesthetic procedures
has fueled novel techniques and has defined new trends
in BoNT-A treatment in Asians. However, to further
refine the techniques and clarify any safety issues, the
need to establish concrete scientific evidence for these
novel indications is paramount.
Further reading
Carruthers A, Carruthers J 1998 Clinical indications and injection
technique for the cosmetic use of botulinum A exotoxin.
Dermatologic Surgery 24(11):1244–12447
Carruthers A, Carruthers J 2008 Botulinum toxin products
overview. Skin Therapy Letter 13(6):1–4
Carruthers J, Fagien S, Matarasso SL 2004 Botox consensus group.
Consensus recommendations on the use of botulinum toxin
type A in facial aesthetics. Plastic and Reconstructive Surgery
114(6 suppl):1S–22S
Carruthers J, Glogau RG, Blitzer A 2008 Facial Aesthetic
Consensus Group Faculty. Advances in facial rejuvenation:
botulinum toxin type A, hyaluronic acid dermal fillers, and
combination therapies – consensus recommendations. Plastic
and Reconstructive Surgery 121(5 suppl):5S–30S
Chang SP, Tsai HH, Chen WY, et al 2008 The wrinkles soothing
effect on the middle and lower face by intradermal injection of
botulinum toxin type A. International Journal of Dermatology
47:1287–1294
Dessy LA, Mazzocchi M, Rubino C, et al 2007 An objective
assessment of botulinum toxin A effect on superficial skin
texture. Annals of Plastic Surgery 58:469–473
Kim K, Shin H-I, Kwon BS, et al 2011 Neuronox versus BOTOX
for spastic equinus gait in children with cerebral palsy: a
randomized, double-blinded, controlled multicentre clinical trial.
Developmental Medicine and Child Neurology 53(3):239–244
Disuse muscular atrophy induced by BoNT-A could be
applied to other areas such as the temple, calves and upper
arms (see Table 8.2). Hypertrophied temporalis muscle
may result in a less attractive bulging appearance, and
BoNT-A can create smooth and neat contours by volume
reduction of temporalis muscle. Calf muscle reduction has
rapidly become popular in Asia, as a close second to mas­
seter reduction in terms of muscle volume reduction by
BoNT-A. Patients are more keen on reducing the muscular
bulge during walking or in tip-toe position than in the
diameter itself. Reducing muscle bulge makes the lower
legs look slimmer (Fig. 8.5). Shaping the upper arms by
reducing deltoid muscle is another novel indication of
BoNT-A utilizing disuse atrophy. With 50 Unit per side,
it can deliver impressive results and patient satisfaction
(Fig. 8.6).
Figure 8.6  Upper arm became slender after
injection of BoNT-A (50 unit per side) into
deltoid muscle: above, pretreatment; below,
2 months post-treatment.
Pearl 3 
Unnatural bulging of focal muscle upon mastication may also
occur after treatment of masseter hypertrophy with BoNT-A.
This is caused by the imbalance of muscle activity between
the muscle fibers affected and those unaffected by BoNT-A.
This symptom can be a serious concern because it looks
weird to others. This side effect usually begins at 1–3 days
after treatment and resolves spontaneously within a month,
but an additional injection into the upper or deeper part of
the masseter muscle unaffected by the initial treatment can
help correct this embarrassing adverse effect more quickly.

58
Botulinum toxin
Kim HJ, Yum KW, Lee SS, et al 2003 Effects of botulinum toxin
type A on bilateral masseteric hypertrophy evaluated with
computed tomographic measurement. Dermatologic Surgery 29:
484–489
Lietzow MA, Gielow ET, Le D, et al 2008 Subunit stoichiometry
of the Clostridium botulinum type A neurotoxin complex
determined using denaturing capillary electrophoresis. Protein
Journal 27(7-8):420–425
Rho NK, Kim HS, Kim YS, et al 2010 Botulinum toxin type A for
facial wrinkles and benign masseter hypertrophy in Korean
patients. Korean Journal of Dermatology 48(10):823–831
Seo KK 2005 Clinical tips and recent advances in cosmetic uses of
botulinum toxin including mesobotox. Journal of the Korean
Medical Association 48:1225–1232
Shim WH, Yoon SH, Park JH, et al 2010 Effect of botulinum toxin
type A injection on lower facial contouring evaluated using a
three-dimensional laser scan. Dermatologic Surgery 36
(suppl 4):2161–2166
Smyth AG. 1994 Botulinum toxin treatment of bilateral masseteric
hypertrophy. British Journal of Oral and Maxillofacial Surgery
32(1):29–33
Stone AV, Ma J, Whitlock PW, et al 2007 Effects of Botox and
Neuronox on muscle force generation in mice. Journal of
Orthopaedic Research 25(12):1658–1664
Xu JA, Yuasa K, Yoshiura K, et al 1994 Quantitative analysis
of masticatory muscles using computed tomography.
Dentomaxillofacial Radiology 23:154–158
Yoon JS, Kim JC, Lee SY 2009 Double-blind, randomized,
comparative study of Meditoxin versus Botox in the treatment
of essential blepharospasm. Korean Journal of Ophthalmology
23(3):137–141
Zhang L, Lin W-J, Li S, et al 2003 Complete DNA sequences of
the botulinum neurotoxin complex of Clostridium botulinum
type A-Hall (Allergan) strain. Gene 315:21–32

Introduction
Botulinum toxin is a naturally occurring protein produced
by the anaerobic bacterium Clostridium botulinum. The
protein acts as a neurotoxin by blocking the release of
acetylcholine at the neuromuscular junction of striated
muscle, thereby blocking neuromuscular motor transmis­
sion. This unique property has been found to have great
clinical utility, treating such diverse medical conditions as
blepharospasm, hyperhidrosis, and dynamic rhytides.
Seven serologically distinct types of botulinum toxin
have been identified – designated as types A, B, C1, D, E,
F, and G. Only botulinum toxin A and B are used clinically,
with serotype A being the most potent. At this time,
rimabotulinumtoxinB or Myobloc® (Solstice Neurosci­
ences, Inc; South San Francisco, CA) is the only com­
mercially available botulinum toxin B. Initially approved
by the FDA in 2000 for the treatment of cervical dystonia,
its use in cosmetic applications has been limited by sys­
temic anticholinergic adverse effects and high antigenicity.
Therefore the following discussion will focus solely on
botulinum toxin A (BoNT-A).
BoNT-A, like all the botulinum toxin serotypes, causes
flaccid muscle paralysis by blocking acetylcholine release
at neuromuscular junctions. The compound, first purified
in 1946 by Dr Edward Shantz, was first found to have a
cosmetic application in 1988 when Drs Jean and Alastair
Carruthers observed improvement in the appearance of
periorbital rhytides in patients treated for blepharospasm.
In 1992, the Carruthers published their first study describ­
ing its cosmetic use in the treatment of glabellar frown
lines. Subsequent randomized controlled studies provided
evidence for its safety and efficacy leading to FDA approval
of BoNT-A for cosmetic procedures in 2003. Today
BoNT-A is considered a vital component of non-invasive
facial rejuvenation and is the most common non-surgical
cosmetic procedure performed.
Types of botulinum toxin A
There are multiple formulations of BoNT-A available in
the US and internationally; however, the practitioner must
9 
Comparison of botulinum toxins
Chung-Yin Stanley Chan, Kathryn Kent,
Thomas E. Rohrer
Summary and Key Features
•
Botox® (onabotulinumtoxinA), Dysport®
(abobotulinumtoxinA) and Xeomin®
(incobotulinumtoxinA) are the three commercially
available formulations of BoNT-A existing in the US
today
•
All three formulations are FDA approved for the
treatment of glabellar lines; in addition, all are
highly effective in treatment of dynamic rhytides of
the face and neck
•
The botulinum neurotoxin molecule is a single-
chain polypeptide found in its native state
surrounded by varying amounts of complexing
proteins; Botox® contains a larger number of
complexing proteins than Dysport®; Xeomin® does
not contain complexing proteins
•
Although the exact dosing ratio is unclear and may
vary depending on application, the ratio between
Botox® : Dysport® : Xeomin® is likely to be around
1 : 2.5 : 1
•
Clinically the duration of action appears to
be similar for all currently available BoNT-A
preparations; however, further investigation is
needed
•
Some evidence suggests that the degree of
diffusion may vary between formulations when
used to treat hyperhidrosis; however, these
differences, if any, are subtle
•
Therapeutic failure of BoNT-A in cosmetic use
is very rare and the role of blocking antibodies
remains unclear
•
The safety of Botox® and Dysport® has been well
established and Xeomin®, while a newer product,
appears to have a similar safety profile
•
Vials of Botox® and Dysport® must be kept
refrigerated prior to reconstitution; however,
Xeomin® may be stored at room temperature prior
to use

60
Botulinum toxin
be aware that these products are not interchangeable and
important differences exist. As such, in 2009 the US FDA
established unique drug names to ‘help ensure patient
safety and reduce confusion’ (Table 9.1). What follows is
a review of available products and their known and theo­
retical differences.
There are currently three formulations of BoNT-A
available in the US today for cosmetic applications: ona­
botulinumtoxinA or Botox® (Allergan, Inc, Irvine, Cali­
fornia, USA), abobotulinumtoxinA or Dysport® (Ipsen
Biofarm Ltd, Berkshire, UK) and incobotulinumtoxinA or
Xeomin®® (Merz Pharmaceuticals, Frankfurt am Main,
Germany). For ease of discussion, the products will be
referenced by their trade names moving forward. Botox®
(Fig. 9.1) was the first to become available and continues
to dominate the worldwide cosmetic market. In the USA
it is approved for the treatment of glabellar lines and
hyperhidrosis. It has also been shown to be highly effec­
tive for such ‘off-label’ uses as the treatment of dynamic
rhytides of the forehead, periorbital area, midface, peri­
oral area, and neck.
Dysport® (Fig. 9.2), another preparation of BoNT-A, is
approved for the treatment of glabellar lines. It has
become increasingly popular worldwide since its introduc­
tion and has been shown to have similar cosmetic applica­
tions to Botox®.
A third product, Xeomin® (Merz Pharmaceuticals,
Frankfurt am Main, Germany) was previously indicated
only for treatment of cervical dystonia and blepharospasm.
However, as of July 2011, it also gained FDA approval for
Table 9.1  Comparison of the currently US-approved botulinum toxin A products
OnabotulinumtoxinA
AbobotulinumtoxinA
IncobotulinumtoxinA
RimabotulinumtoxinB
US Tradename
Botox®
Dysport®
Xeomin®
Myobloc®
Company
Allergan, Inc.
Ipsen Inc. / Medicis
Merz Pharmaceuticals
Solstice Neurosciences
Inc. / Eisai Co., Ltd.
Active substance
BoNT-A complex
BoNT-A complex
BoNT-A free from
complexing proteins
BoNT-B complex
Molecular weight
900 kDa
500–900 kDa
150 kDa
700 kDa
Target protein
SNAP-25
SNAP-25
SNAP-25
VAMP
Units per vial
50 or 100
300 or 500
100
2500, 5000, or 10 000
Pharmaceutical form
Powder
Powder
Powder
Solution
US FDA-approved
indications
Blepharospasm, cervical
dystonia, glabellar lines,
hyperhidrosis, chronic
migraine
Blepharospasm, cervical
dystonia, glabellar lines
Blepharospasm,
cervical dystonia,
glabellar lines
Cervical dystonia
Storage temperature
before and after
reconstitution
2–8°C / 2–8°C
2–8°C / 2–8°C
<25°C / 2–8°C
2–8°C / 2–8°C
Figure 9.1  Vial of Botox, botulinum toxin A with the unreconstituted
product seen as the small white ring of powder visible around the
base of the vial. Each vial contains 100 units of botulinum exotoxin A
in lyophilized form, as well as a small amount of sodium chloride
(0.9 mg) and human albumin (0.5 mg).

61
9 
Comparison of botulinum toxins
Mechanism of action
All botulinum toxin serotypes produce their clinical
effect by blocking neuromuscular transmission. Specifi­
cally, BoNT-A acts through the cleavage of SNAP-25
(synaptosomal-associated protein of 25 kDa), a protein
required for the release of acetylcholine at the neuro­
muscular junction. The toxin anchors to motoneuronal
synapses and becomes internalized via its heavy chain.
The light chain of the toxin then catalyzes the cleavage
of SNAP-25 with the resultant inhibition of acetylcho­
line release. All BoNT-A products act through this
mechanism.
Compositional differences
The botulinum neurotoxin molecule is a 150 kDa single-
chain polypeptide consisting of a 100 kDa heavy chain and
a 50 kDa light chain. These chains are held together by a
disulfide bond and are associated with a zinc atom (Fig.
9.3). In its native state the 150 kDa toxin is found com­
plexed with large protective proteins. These complexing
proteins, which are primarily hemagluttinins, act to protect
ingested toxin from degradation within the gastrointestinal
tract. In neutral to basic pH environments the BoNT-A
complex dissociates into the free 150 kDa neurotoxin and
high-molecular-weight hemagglutinin components.
The complexing proteins of the native molecule seem
to have evolved as a clever mechanism to protect the toxin
when ingested by the host. However, the role that these
proteins play, if any, in cutaneous injection of BoNT-A is
unclear and remains a subject of debate. It has been
argued that the proteins help to stabilize the core toxin or
that they may help to enhance activity; however, these
hypotheses have not been substantiated.
The three available formulations of BoNT-A vary in the
presence, absence, and amount of complexing proteins.
Botox® is the largest of the available preparations at
900 kDA (Fig. 9.4) and is associated with the most com­
plexing proteins. Due to the method employed in its
production, Dysport® may be associated with a variable
number of proteins with a molecular weight ranging from
500 to 900 kDa. Uniquely in the group, Xeomin® is com­
posed only of the 150 kDa neurotoxin, free of any com­
plexing proteins. Purtox® will also be a non-complexed
formulation of botulinum toxin A.
There is good evidence to support the concept that the
complexing proteins in Botox® and Dysport® may dissoci­
ate when the vial is reconstituted with saline. A recent
study by Eisele et al (funded by Merz GmbH) showed
that, after reconstitution by manufacturer standards, over
85% of the Botox® was present in the free state without
associated hemagluttinins. The remainder of the neuro­
toxin was associated with complexes in the 500 kDa range
as opposed to its original 900 kDa complex. In this study,
Dysport® was found to be only in the free neurotoxin
state. Furthermore, the complexing proteins dissociate
readily after being injected into the higher pH of the skin.
the treatment of glabellar rhytides. Xeomin® has become
available in the United States in the spring of 2012.
Other BoNT-A products currently in development
include Purtox® (Mentor Corporation, Santa Barbara,
California, USA), Neuronox® (Medy-Tox, Chung-cheongbuk-
do, Korea), and Prosigne® (Lanzhou, China). Purtox®
recently completed the first of three Phase III clinical
trials with promising results. Clinical trial data in the
United States are still pending on the other BoNT-A for­
mulations, some of which are currently available outside
the United States. In the future it will be interesting to
see how the addition of other BoNT-A products to the
market will affect product pricing and patient access to
them.
Figure 9.2  Vial of Dysport® supplied as sterile, single-use, 3 mL
glass vial. Each vial contains either 300 or 500 units of lyophilized
form of the botulinum toxin type.

62
Botulinum toxin
amount of BoNT-A used is so minute that it must be mea­
sured in terms of its biologic activity rather than by its
mass. The dose of BoNT-A, usually in the nanogram range
(10−9), is measured in units of biologic activity in a stan­
dardized mouse model. One unit of biological activity,
commonly referred to as 1 unit, is the amount of toxin
required to kill 50% (lethal dose or LD50) of 18–20 g female
Swiss-Webster mice when injected intraperitoneally. For
comparison, the estimated lethal dose (LD50) in a 100 kg
human is approximately 3500 times this, or 3500 units.
For the unit defined by the mouse model, 1 unit of
Botox® will have similar potency to 1 unit of Dysport®
and to 1 unit of Xeomin®. However, there is significant
interspecies variation and it has been shown that this same
one-to-one relationship does not hold true for Dysport®
when compared with Botox® and Xeomin® in other
species. Further muddying the waters is the way in which
early studies measuring the LD50 of the different formula­
tions were designed. The diluents used in assays measuring
the LD50 of the products were not identical. Dysport®
In this study, the proteins are estimated to dissociate in
less than 60 seconds. Therefore, the small number of
complexing proteins remaining after reconstitution seem
to dissociate rapidly upon injection into the skin. This
argues against the complexing proteins having any effect
on the spread or diffusion of the toxin. The presence of
complexing proteins may, however, contribute to BoNT-A
resistance, as discussed later.
Dosing
It is imperative that the practitioner be knowledgeable
regarding the differences in dosing for the available
BoNT-A formulations. Unfortunately, there is currently
no accepted ‘conversion factor’ between products, and
this remains an area of controversy and debate. To appreci­
ate product-dosing differences, an understanding of the
definition of unit potency is helpful.
Most drugs are measured and administered by their
weight, typically in the milligram range. However, the
Figure 9.3  The structure of the 150 kDa botulinum toxin A: it is composed of a 100 kDa heavy chain (responsible for the high-affinity,
irreversible, docking on the presynaptic nerve membrane) on the left and a 50 kDa light chain (responsible for the cleavage of proteins in
the synaptic fusion complex) on the right. The heavy and light chains are held together by a disulfide bond.
Binding
domain
Translocation
domain
Catalytic domain
Translocation belt
50 loop
200 loop
250 loop

63
9 
Comparison of botulinum toxins
Efficacy
Trials on efficacy are somewhat difficult to interpret given
the lack of standardized dosing between the products as
discussed above. Assuming appropriate and equivalent
dosing ratios, there is no evidence to suggest greater effi­
cacy of any of the BoNT-A preparations. All have been
shown to be efficacious in treating glabellar rhytides, gar­
nering their FDA indication for this treatment. They are
also used ‘off-label’ for other indications with comparable
results.
toxin was diluted by a phosphate buffer containing gelatin
that stabilizes low-concentration toxins, whereas saline
was used in the Botox® assays leading to apparently
reduced potency. Though experience with Xeomin®
remains limited, clinical studies have demonstrated a dose
ratio of roughly 1 : 1 when compared with Botox®.
Clinical studies have failed to yield a clear consensus
concerning the precise dose ratio between the BoNT-A
preparations as standardization of clinical studies has been
lacking. One review by Karsai & Raulin comparing Botox®
and Dysport® showed that a dose ratio of less than 1 : 3 is
probably most suitable for the two products. More specifi­
cally, others have proposed a dose ratio of 1 : 2.5, whereas
the largest retrospective review on the topic by Hevia
established a rather cumbersome dose ratio of 1 : 2.67. In
our practice, we believe a dosing ratio of 1 : 2.5 between
Botox® and Dysport® is accurate.
The conversion ratio between Botox® and Xeomin® is
also not well established. However, in two large compara­
tive trials by Benecke and colleagues using a conversion
factor of 1 : 1, non-inferiority of Xeomin® to Botox® was
shown, establishing the likelihood of similar dosing
between the two products. Although the precise dosing
of the BoNT-A preparations is by no means agreed upon,
in practice the dosing ratio between Botox® : Dysport® : 
Xeomin® is likely to be around 1 : 2.5 : 1.
Figure 9.4  The structure of the large 900 kDa botulinum toxin A:
note that the actual botulinum toxin itself makes up only 150 kDa of
the structure; the rest is made up of large protective hemagglutinin
(HA) and non-toxic non-hemagglutinin (NTNH) proteins. The complex
is held together by non-covalent bonds that are stable in the acidic
environment of the gastrointestinal tract, but dissociate as the pH
increases in the blood stream and releases the free toxin. (LC = light
chain; HC = heavy chain; S–S = disulfide bond; Zn = zinc.)
S
HA
HA
HA
HA
HA
HA
Zn
HC
LC
NTNH
S
Pearl 2 
Although botulinum toxin in cosmetics is currently FDA
approved only for glabellar rhytides and hyperhidrosis, its
scope of use is much broader, with applications in treating
other facial rhytides and conditions.
Pearl 1 
The clinical dosing ratio between Botox® : Dysport® : Xeomin®
is approximately 1 : 2.5 : 1.
Duration of action
The precise duration of action of BoNT-A is difficult to
assess and is dependent on a host of factors including
the area treated, patient characteristics, and dosing. The
currently available preparations appear to have a lasting
effect of 3–4 months when employed for aesthetic use.
In general, there is a trend towards a longer duration of
action when higher dosages are used, up to a plateau point.
Furthermore, when skeletal muscle is repeatedly treated
with BoNT-A, some degree of atrophy may be demon­
strated. This may result in future treatments that require
less units of toxin as well as treatments that may occur at
increased time intervals. It is these authors’ observation
that patients retain some improvement of their dynamic
rhytides for more than a year even without repeat treat­
ment. Patients returning 1–2 years following a single
BoNT-A injection often have less apparent rhytides by
photographic evaluation when compared with before their
initial treatment. One explanation for this is that, because
there is less collagen at the depth of an active rhytide,
allowing the area to rest for a period of time may give the
body the opportunity to deposit more collagen in the
depth of the rhytide.
The existing comparative trials on duration of action
between Botox® and Dysport® vary across a wide range of
doses making interpretation difficult; however, the pre­
ponderance of data suggest that the two have similar dura­
tions of action. A recent study by Jost and colleagues
comparing the effects of treatment with Xeomin® and
with Botox® on muscle activity of the hand showed both
products to have a similar duration of effect. Until further

64
Botulinum toxin
profiles of Botox® and Dysport® (reviewed by De Baulle
et al). Although Xeomin® is a newer BoNT-A with fewer
clinical studies than the others, it was shown by Wabbels
and colleagues to have a similar safety profile as Botox®.
When adverse events related to injection do occur
they are usually transient and mild in severity and most
often arise when the injector is inexperienced or has
poor technique. The two most common adverse events
encountered with BoNT-A injection are headache and
eyelid ptosis, both being more common in those being
injected for the first time. The long-term safety of com­
plexing proteins has not been studied specifically, but is
unlikely to have any clinical significance given the overall
safety of BoNT-A. Data on comparative immunogenicity
of the products are also not available. To help maintain
patient safety and optimize outcomes it is vital for the
practitioner to possess a comprehensive understanding
of the physiology and the properties of each BoNT-A
formulation.
Resistance
Therapeutic failure of BoNT-A in cosmetic use is very
uncommon and has been reported in only a few instances.
It is believed that the development of resistance to treat­
ment is due to the production of antibodies directed
against both the heavy and light chain components of the
neurotoxin. The precise cause of antibody development is
rather complex and remains to be elucidated. However,
as with other immunologic events, the production of anti­
bodies depends on the immune system of the host as well
as the immunogenicity of the stimulus. The external
factors that appear to be most associated with the devel­
opment of blocking antibodies are the dose per session
and the interval between doses. The cumulative dose does
not seem to be as important. Patients treated with high
doses (300 units of Botox® or higher) at frequent inter­
vals, as in the neurological setting, seem the most likely to
develop antibodies. Cosmetic patients treated with
Botox® typically utilize 25–75 units at intervals of several
months and are extremely unlikely candidates for devel­
oping resistance.
The older, functional Botox® literature reports blocking
antibodies in approximately 5% of patients treated for
cervical dystonia. However, these patients were treated
with the original lot of Botox®, which had a much higher
protein content (25 ng/vial) than does the present lot
(5 ng/vial). Whereas reports of antibody formation with
BoNT-A are more common in neurological patients, the
significantly lower dosages and subsequently smaller
protein loads used in cosmetics have made this scenario
exceedingly rare.
Although few may develop these blocking antibodies,
patient non-response to treatment with BoNT-A occurs
even less frequently. This is because patients who develop
antibodies may still have a clinical response to BoNT-A.
According to Allergan, only 1–2% of patients will develop
antibodies to Botox® and the correlation of these
studies are performed it is reasonable to expect that the
different BoNT-A products will not differ significantly in
their duration of action.
Diffusion / field of effect
Initially it was hypothesized that variations in the number
of complexing proteins found in the BoNT-A complex
would alter the degree of diffusion of a given product.
Formulations containing a greater amount of complexing
proteins with resultant higher molecular weights were
speculated to have decreased areas of diffusion due to
particle size. Additionally, a pilot study by Trindade and
co-workers comparing the degree of diffusion of Dysport®
and Botox® on forehead hyperhidrosis showed that
Dysport®, with a lower molecular weight, had a greater
area of diffusion as evidenced by the size of the anhidrotic
halo produced. The clinical significance of this finding was
less clear, however, as there was no relationship between
anhidrosis and inhibition of frontalis muscle contraction.
Newer studies have shown the diffusion between the
products to be similar and have cast doubt on the role of
complexing proteins in the diffusion of toxin. In a mouse
model using a highly sensitive assay to measure diffusion,
Botox®, Dysport®, and Xeomin® all had similar degrees of
diffusion. Since most toxin exists in its unbound form
after reconstitution, it is logical that complexing proteins
would have little effect on area of diffusion. Nevertheless,
until further definitive studies are performed one should
be mindful of the possible diffusion differences between
formulations.
One factor that has been shown to affect the diffusion
of BoNT-A is the dilution used. BoNT-A is dispensed in a
crystallized form that must be reconstituted with saline
before use. The amount of saline used to reconstitute the
product, which may vary by physician preference, will
affect its diffusion. A study by Hsu et al comparing Botox®
mixtures reconstituted with 1 mL and 5 mL of saline
found a 50% greater distance of diffusion in the more
dilute mixture. This property may be used to the physi­
cian’s advantage to create larger field effects. Conversely,
for the uneducated practitioner, more extensive diffusion
may potentially result in unintended consequences such
as lid ptosis.
Pearl 3 
Reconstitution with larger volumes of saline has been shown
to result in greater diffusion of the product, which can be
used to the clinician’s advantage. Care must be taken to
account for this diffusion to reduce unwanted side effects.
Safety
The safety of BoNT-A in cosmetic use has been well
established in the literature and the increasing popularity
of such procedures is also a testament to this. Both short-
and long-term studies exist supporting the favorable safety

65
9 
Comparison of botulinum toxins
Pearl 4 
Therapeutic failure of botulinum toxin in cosmetic use is
extremely uncommon. A trial of a different subtype of toxin
(botulinum toxin B) may be considered.
available BoNT-A products used today. Understanding the
compositional differences of the products allows for more
detailed and precise injection techniques with limited
side effects. Fortunately, the currently available products
appear to have similar efficacy, safety profiles and duration
of action, but the role of complexing proteins still merits
further investigation. Moving forward, it will be important
to stay familiar with evolving toxin products.
Further reading
Benecke R, Jost WH, Kanovsky P, et al 2005 A new botulinum
toxin type A free of complexing proteins for treatment of
cervical dystonia. Neurology 64(11):1949–1951
Binder WJ 2006 Long-term effects of botulinum toxin type A
(Botox) on facial lines: a comparison in identical twins. Archives
of Facial Plastic Surgery 8(6):426–431
Brin MF 2009 Basic and clinical aspects of BOTOX. Toxicon
54(5):676–682
Carli L, Montecucco C, Rossetto O 2009 Assay of diffusion of
different botulinum neurotoxin type a formulations injected in
the mouse leg. Muscle Nerve 40:374–380
Carruthers A, Carruthers J 1998 History of cosmetic use of
botulinum A exotoxin. Dermatologic Surgery 24:1168–1170
Carruthers A, Carruthers J 2005 Botulinum toxin type A. Journal
of the American Academy of Dermatology 53(2):284–290
Carruthers JA, Lowe NJ, Menter MA, et al 2002 A multicentre,
double-blind, randomized, placebo-controlled study of efficacy
and safety of botulinum toxin type A in the treatment of
glabellar lines. Journal of the American Academy of
Dermatology 46:840–849
Carruthers JD, Carruthers JA 1992 Treatment of glabellar frown
lines with C. botulinum-A exotoxin. Journal of Dermatologic
Surgery and Oncology 18:17–21
De Boulle K, Fagien S, Sommer B, et al 2010 Treating glabellar
lines with botulinum toxin type A-hemagglutinin complex: a
review of the science, the clinical data, and patient satisfaction.
Clinical Interventions in Aging 5:101–118
Dressler D 2004 Clinical presentation and management of antibody
induced failure of botulinum toxin therapy. Movement
Disorders 19(8 suppl):S92–S100
Dressler D, Eleopra R 2006 Clinical use of non-A botulinum toxins:
botulinum toxin type B. Neurotoxicity Research 9(2-3):121–125
Dressler D, Wohlfahrt K, Meyer-Rogge E, et al 2010 Antibody
induced failure of botulinum toxin a therapy in cosmetic
indications. Dermatologic Surgery 36(suppl 4):2182–2187
Eisele KH, Fink K, Vey M, et al 2011 Studies on the dissociation
of botulinum neurotoxin type A complexes. Toxicon 57(4):555–
565
Flynn TC 2010 Botulinum toxin: examining duration of effect in
facial aesthetic applications. American Journal of Clinical
Dermatology 11(3):183–199
Hevia O 2010 Retrospective review of 500 patients treated with
abobotulinumtoxin A. Journal of Drugs in Dermatology
9:1081–1084
Hsu TS, Dover JS, Arndt KA 2004 Effect of volume and
concentration on the diffusion of botulinum exotoxin A.
Archives of Dermatology 140(11):1351–1354
Inoue K, Fujinaga Y, Watanabe, et al 1996 Molecular composition of
Clostridium botulinum type A progenitor toxins. Infection and
Immunity 64:1589–1594
Jost WH, Kohl A, Brinkmann S, et al 2005 Efficacy and tolerability
of a botulinum toxin type A free of complexing proteins (NT
201) compared with commercially available botulinum toxin
type A (BOTOX) in healthy volunteers. Journal of Neural
Transmission 112(7):905–913
antibodies to clinical response is not clear (see the 2010
analysis by Naumann and colleagues).
Therapeutic failure due to autoantibodies has been
reported for both Dysport® and Botox® in cosmetic use.
There are currently no reports of antibody-induced failure
due to Xeomin®. Whether this is secondary to the lack of
experience with the product or the lack of complexing
proteins remains to be seen.
Conclusion
As BoNT-A injections have become the most common
non-invasive cosmetic procedure performed in this country,
it is vital to appreciate the differences between the
For patients who are resistant to chemodenervation
with one serotype of BoNT, a trial with a different sero­
type is indicated. For example, a patient that has devel­
oped a resistance to one of the BoNT-A preparations
should have a trial with the BoNT-B preparation. In addi­
tion, an attempt should be made to measure antibodies to
determine whether the resistance is antibody mediated.
The newer BoNT-A products should be further evaluated
for antibody formation rates as their protein load is
different.
Storage
Vials of Botox® had traditionally been kept frozen (−5oC)
until used. However, new evidence suggests that there is
no significant change in the shelf life or efficacy of Botox®
if it is stored in a refrigerator rather than a freezer. The
package insert now recommends that the vials be stored
at 2–8oC. Similarly, vials of Dysport® may be kept refriger­
ated as opposed to frozen prior to use. One potential
advantage of Xeomin® is that it can be stored at room
temperature for up to 36 months. It is noteworthy that
all of the commercially available neurotoxins bear labels
indicating that they should be discarded if the contents
are not used within 4 hours of reconstitution / broaching
the vial. This is in accordance with FDA regulations;
however, studies by Trindade et al have shown there to be
no decrease in product efficacy when stored for weeks to
months in refrigeration.
Pearl 5 
All commercially available formulations bear labels indicating
they should be discarded if not used within 4 hours of
reconstitution. However, studies have shown no decrease in
efficacy weeks to months after reconstitution providing the
product remains refrigerated.

66
Botulinum toxin
Karsai S, Raulin C 2009 Current evidence on the unit equivalence
of different botulinum neurotoxin A formulations and
recommendations for clinical practice in dermatology.
Dermatologic Surgery 35(1):1–8
Karsai S, Raulin C 2010 Botox and Dysport: is there a dose
conversion ratio in dermatology and aesthetic medicine? Journal
of the American Academy of Dermatology 62(2):346–347
Lee SK 2007 Antibody-induced failure of botulinum toxin type A
therapy in a patient with masseteric hypertrophy. Dermatologic
Surgery 33(1 spec. no.):S105–S110
Meunier FA, Schiavo G, Molgo J 2002 Botulinum neurotoxins:
from paralysis to recovery of functional neuromuscular
transmission. Journal of Physiology (Paris) 96:105–113
Middlebrook JL 1989 Cell surface receptors for protein toxins.
In: Simpson LL (ed) Botulinum neurotoxin and tetanus toxin.
Academic Press, San Diego, p 95–119
Naumann M, Carruthers A, Carruthers J, et al 2010 Meta-analysis
of neutralizing antibody conversion with onabotulinumtoxinA
(BOTOX(r)) across multiple indications. Movement Disorders
25(13):2211–2218
Roggenkämper P, Jost WH, Bihari K, et al 2006 Efficacy and safety
of a new Botulinum Toxin Type A free of complexing proteins in
the treatment of blepharospasm. Journal of Neural Transmission
113(3):303–312
Sattler G 2010 Current and future botulinum neurotoxin type A
preparations in aesthetics: a literature review. Journal of Drugs
in Dermatology 9(9):1065–1071
Schiavo G, Matteoli M, Montecucco C 2000 Neurotoxins affecting
neuroexocytosis. Physiological Reviews 80:717–766
Sharma SK, Singh BR 2004 Enhancement of the endopeptidase
activity of purified botulinum neurotoxins A and E by an
isolated component of the native neurotoxin associated proteins.
Biochemistry 43:4791–4798
Simpson LL 1986 Molecular pharmacology of botulinum toxin and
tetanus toxin. Annual Review of Pharmacology and Toxicology
26:427–453
Trindade de Almeida AR, Marques E, et al 2007 Pilot study
comparing the diffusion of two formulations of botulinum toxin
type A in patients with forehead hyperhidrosis. Dermatologic
Surgery 33(1 spec. no.):S37–S43
Trindade DE Almeida AR, Secco LC, et al 2011 Handling
botulinum toxins: an updated literature review. Dermatologic
Surgery 37(11):1553–1565
Tsai YC, Maditz R, Kuo CL, et al 2010 Targeting botulinum
neurotoxin persistence by the ubiquitin–proteasome system.
Proceedings of the National Academy of Sciences USA
107:16554–16559
Wabbels B, Reichel G, Fulford-Smith A, et al 2011 Double-blind,
randomised, parallel group pilot study comparing two botulinum
toxin type A products for the treatment of blepharospasm.
Journal of Neural Transmission 118(2):233–239

aesthetic medicine. In 2002, the United States (US) FDA
approved the use of Botox® Cosmetic (botulinum toxin
type A purified toxin complex; Allergan, Inc.) for the
treatment of moderate-to-severe glabellar rhytides, associ­
ated with corrugator and / or procerus muscle activity
in adult patients 65 years of age and younger. In 2004,
Carruthers et al reported the consensus recommendations
that BoNT-A is effective and safe, and patient satisfaction
is high. By 2005, Botox® cosmetic injections were the
most common non-invasive physician-administered cos­
metic procedure worldwide.
Need for transcutaneous delivery systems
Matarasso & Matarasso reported in 2001 that the paralytic
effect of injected BoNT-A can span up to 3 cm from the
site of injection and even further when dilute concentra­
tions and large volumes of BoNT-A are used. Limitations
of injections of BoNT-A include pain, erythema, swelling,
potential infection from needle use, and potential for
reduced normal expression in the treated area. The
patient’s pre-treatment medical regimen is potentially
impacted by being advised to avoid aspirin, non-steroidal
anti-inflammatory drugs, and vitamin E prior to injection,
to reduce the risk of bleeding and bruising. Bruising is of
particular concern in the crow’s feet (also known as lateral
canthal lines or LCL), where the blood vessels are super­
ficial and the skin is thin. Because of the disadvantage of
requiring injection as the route of administration, alterna­
tive methods of drug delivery have been developed that
can address some of these concerns.
Conventional transepidermal drug delivery systems can
deliver only small molecules. Some examples of smaller
molecules that can be delivered through the skin topically
include progesterone, scopalomine, certain antibiotics,
and nicotine. These are readily delivered in traditional
topical preparations, creams, ointments, adhesive patches,
etc. These systems range from very inefficient to com­
pletely ineffective in delivering biologically active proteins
and other macromolecules across the skin barrier. The
stratum corneum and upper layers of the epidermis are
lipid-rich barriers to entry for most of the larger mole­
cules, which is, after all, the skin’s primary function – to
exclude assaults on the inner host by biologically active
macromolecules. The flux of most proteins across the skin
barrier is essentially zero.
10 
Topical neurotoxin
Jacob M. Waugh, Richard G. Glogau
Background
Injectable botulinum toxin type A (BoNT-A) has been
successfully used across a range of medical disorders
including strabismus, blepharospasm, focal dystonias,
migraine, spasticity associated with juvenile cerebral
palsy and adult stroke, and various cosmetic treatments.
Thus, the safety and effectiveness of BoNT-A for treating
these conditions have been well established over the past
20 years.
While aesthetic medicine has been inundated with
treatments for facial rhytides, BoNT-A is the only Food
and Drug Administration (FDA)-approved treatment to
temporarily relax hyperfunctional rhytides. Since 1992,
botulinum toxin has been used to treat a variety of cos­
metic conditions and medical indications. Following the
first description of the treatment for glabellar rhytides
(‘frown line’ wrinkles) by Carruthers & Carruthers in
1992, botulinum toxin has revolutionized the practice of
Summary and Key Features
•
Injectable BoNT-A has been successfully used
across a range of medical disorders including
strabismus, blepharospasm, focal dystonia,
migraine, spasticity associated with juvenile
cerebral palsy and adult stroke as well as various
cosmetic treatments such as to temporarily relax
hyperfunctional rhytides.
•
Alternative methods have been developed due to
limitations and disadvantages of injections of
BoNTA which includes iontophoresis device-based
approaches and topical liposomes and CPP drug
delivery systems.
•
Topical botulinum toxin delivery through the skin
may offer opportunities to treat anatomical areas
that are either difficult to manage with injectables
or where patients may prefer to avoid injectables,
and there may also be useful roles for topical
botulinum therapies as adjunctive or extender
therapies for the injectable techniques now in use.

68
Botulinum toxin
long-term stability of BoNT-A in their topical prepara­
tions. Neither intends to require reconstitution of a lyoph­
ilized BoNT-A at the time of use. Interestingly, no company
that manufactures BoNT-A has been able to develop a
solution stable form of BoNT-A commercially. Since all
current manufacturers of BoNT-A including all injectable
forms [Allergan’s Botox® Cosmetic (onabotulinum toxin
A); Ipsen’s Dysport® (abobotulinum toxin A); Xeomin®
(incobotulinumtoxinA) from Merz GmbH; Puretox® from
Mentor / J&J in USA; Neuronox® from Medy-tox in
Korea; and Prosigne® / Hengli® from Lanzhou Institute in
China] as well as the investigational topical product
RT001 (Revance Therapeutics in USA) require reconsti­
tution of a stabilized lyophilized BoNT-A, this may prove
a key point for Transdermal Corporation and Anterios.
The remainder of this section will detail progress on
RT001, which is the furthest along in clinical development
of an investigational topical BoNT-A preparation. RT001
is a topical BoNT-A comprised of the 150 kilodalton
(kDA) pure neurotoxin, which is ionically paired with a
CPP-based peptide to afford transepidermal penetration.
There are two distinct yet complementary pathways that
facilitate transcutaneous delivery using a TAT-based CPP
such as that in RT001. The first mechanism is lipid rafting,
which is energy independent and occurs on the non-living
cells of the stratum corneum. The second mechanism is
energy dependent so occurs only across living cells; this
pathway is a transcytosis shuttle, which is a variant of
induced macropinocytosis. This mechanism is the rate-
limiting step for delivery of RT001 so thinning or remov­
ing the stratum corneum appears to have no impact on
drug delivery of this BoNT-A. For macropinocytosis, the
CPP induce the cell to take a large ‘drink’ of the surround­
ings including the CPP. Unlike simple macropinocytosis,
this variant pathway then releases the contents on either
side of the cell. When returned to the original side no net
change occurs, but when returned to the opposite side the
contents have crossed the cell. The result is a net flux
along the concentration gradient across the cells. This
pathway likely takes place on living epithelial cells lining
glands and deeper skin cells, and manifests as a net direc­
tional flux from apical to basolateral surface.
To self-assemble a particle containing both a therapeu­
tic active like botulinum toxin and a CPP-containing
carrier, some form of non-covalent bond must be
employed. For biologically active proteins like BoNT-A,
which often have a conserved motif of high densities of
negatively charged amino acids on their surface, ionic
bonding is particularly facile. RT001 relies on these ionic
interactions to assemble the CPP-containing peptides on
the surface of BoNT-A.
Based upon the initial reports of RT001 by Glogau
and Brandt et al, as well as prior unpublished work, a
full Phase II clinical program for the temporary improve­
ment in the appearance of moderate to severe lateral
canthal lines was completed including over 550 subjects
exposed to RT001. Dose escalation studies were under­
taken across five studies for RT001 in LCL. These studies
Potential approaches to transcutaneous
delivery of BoNT-A
Attempts to increase penetration by manipulation of drug
structure have been largely ineffective for most proteins.
For example, conjugation of a drug to a carrier can com­
promise drug activity and permeation enhancers may
disrupt biological activity of the protein. At present, deliv­
ery of most macromolecules across the skin barrier
requires direct injection (e.g. insulin, antibodies, toxins,
and growth hormone).
Device-based approaches such as iontophoresis have
been explored as well. Iontophoresis utilizes a different
mechanism for drug delivery into the skin, but lacks tar­
geting and delivery specificity and remains time depen­
dent. By utilizing a direct current of relatively low
amplitude, an active electrode is placed on the drug for­
mulation that is to be driven into the skin by the electrons
streaming from the active electrode. The ionic charge
imparted to the target molecule allows the molecule to be
driven into the skin as the indifferent ions are pulled from
the skin by the indifferent electrode to complete the
circuit. This process has typically been used to drive small
charged molecules across skin. Interestingly, iontophoresis
was reported to be successful with botulinum toxin for
treating palmar hyperhidrosis although the practice has
not become widely adopted since the initial reports (see
Kavanagh et al and Solomon). The disadvantages are that
the technique is very time and concentration dependent,
requires that the patient have the ability to tolerate the
sensation of the direct current, and is less effective with
delivery of lipophilic molecules.
Beyond iontophoretic strategies, a number of new
investigational approaches to topical delivery of therapeu­
tics have evolved and several are being evaluated for
BoNT-A. These approaches rely on refinements of conven­
tional topical drug delivery strategies including liposomes
as well as developments in molecular biology including the
past decades of progress in cell-penetrating peptides (CPP,
also known as protein transduction domains or PTDs; see
Chajchir et al). A CPP-based approach for transcutaneous
delivery of BoNT-A is RT001 Topical Gel (Revance Thera­
peutics Inc.). The safety and effectiveness of this approach
have been previously reported from initial studies in both
axillary hyperhidrosis and LCL by Glogau and by Brandt
et al. Chajchir et al had previously reported on microcrys­
tals and ionic nanoparticles for topical BoNT-A in a daily
application (DPM Therapeutics Corp. / Transdermal Cor­
poration). The magnitude of the results from the nanopar­
ticle study was surprising given that the outcome was
achieved with daily topical application of a BoNT-A dose
that would not be expected to attain efficacy when
injected. Recently, clinical trials have also been initiated
to evaluate the safety and effectiveness of liposomes with
a controlled size range to deliver BoNT-A for axillary
hyperhidrosis and LCL (Anterios Inc.).
Although neither Transdermal Corporation nor Ante­
rios are BoNT-A manufacturers, both have described

69
10 
Topical neurotoxin
(1) encompassed all experiences with the proposed Phase
III dose of peptide excipient, (2) employed similar effi­
cacy scales, enrollment criteria, formulation, timepoints,
and consistency of treatment effect, and (3) reflected
planned Phase III studies. All studies that were similar
have been included in this analysis. Escalating doses of
RT001 from 5.5 to 25 ng/mL of BoNT-A led to an
increasing proportion of responders based on 2-point or
greater improvement on a validated investigator global
assessment of lateral canthal line severity (IGA-LCL
scale) whether viewed by individual lateral canthal area
(LCA) per protocol or subject (both eyes responding as
proposed for Phase III studies) as detailed in Table 10.1.
An example is shown in Figure 10.1.
In regard to safety, none of these clinical studies regard­
less of concentration combination revealed any safety
signals of clinical relevance. Specifically, the efficacy
increases observed with escalating doses of RT001 were
not accompanied by dose-dependent increases in adverse
Table 10.1  Response rates by 2 points or greater improvement on IGA-LCL 4 weeks after treatment with RT001
All controls
n = 200
RT001
n = 13
RT001
n = 11
RT001
n = 36
RT001
n = 61
RT001
n = 136
Dosage of BoNT-A
3. 3 ng/mL
5.5 ng/mL
11 ng/mL
22 ng/mL
25 ng/mL
Percentage of subjects
responding in both eyes
8.5%
7.7%
p = 1.0000 [b]
18.2%
p = 0.2592 [b]
22.2%
p = 0.0334 [b]
29.5%
p < 0.0001 [a]
51.5%
p < 0.0001 [a]
Percentage of LCA
responding (n = twice the
number of subjects listed)
9.8%
7.7%
p = 1.0000 [b]
18.2%
p = 0.2637 [b]
26.4%
p < 0.0001 [a]
34.4%
p < 0.0001 [a]
56.3%
p < 0.0001 [a]
Note: The p-value is for the pair-wise comparison of RT001 versus placebo using [a] Pearson’s chi-square test or [b] Fisher’s exact
test.
A
Figure 10.1  A representative 2-point mover in the Phase II trials: (A) baseline; (B) 4 weeks post-treatment. Reproduced with permission of
Revance Therapeutics, Inc.
B
events. Glogau et al reported treatment-emergent adverse
events were generally mild and transient, as in the previ­
ously reported studies by Brandt et al. Local effects
included neuromuscular events directly related to RT001
action on its target muscle, as well as generally mild and
transient eye and skin events that were not dose related.
Safety assessments of cranial nerve evaluation, skin and
ocular irritation, clinical laboratories tests, and ECG
showed no significant treatment or dose related findings.
No evidence of diffusion to adjacent muscles has been
seen with RT001. No evidence of systemic spread or
systemic safety issues was observed in any study. There
were no treatment-related increases in antibody titers to
the neurotoxin or the peptide compared with pre-dose
serum samples. Given this safety profile, reducing the
dose of either RT001 component or both components
could not improve the product’s overall safety profile and
reducing the concentrations of one or both components
would likely decrease efficacy. Any dose reduction would

70
Botulinum toxin
outcome measure, and measures of patient satisfaction
appear to indicate reproducible patient acceptance of the
technique.
Topical botulinum toxin delivery through the skin may
offer opportunities to treat anatomical areas that are
either difficult to manage with injectables or where
patients may prefer to avoid injectables (e.g. the upper lip
rhytides, lateral canthal lines, the frontalis horizontal lines,
the platysma bands, palmar surfaces, scalp, and lower
facial hyperhidrosis). There may also be useful roles for
topical botulinum therapies as adjunctive or extender
therapies for the injectable techniques now in use. Topical
BoNT-A may prove relevant for other conditions such as
orthopedic and ENT indications. Additionally, investiga­
tional use of RT001 for migraine has recently been initi­
ated in Singapore since migraine and other pain indications
may prove amenable to a topical approach as well.
Further reading
American Society for Aesthetic Plastic Surgery, Cosmetic Surgery
National Data Bank 2006 statistics. Online. Available:
30 March 2007
Brandt F, O’Connell C, Cazzaniga A, et al 2010 Efficacy and safety
evaluation of a novel botulinum toxin topical gel for the
treatment of moderate to severe lateral canthal lines.
Dermatologic Surgery 36(suppl 4):2111–2118
Carruthers A, Carruthers J 2005 Botulinum toxin type A. Journal
of the American Academy of Dermatology 53:284–290
Carruthers J, Carruthers A 1992 Treatment of glabellar frown lines
with C. botulinum-A exotoxin. Journal of Dermatologic Surgery
and Oncology 18:17–21
Carruthers J, Carruthers A 2007 The evolution of botulinum toxin
type A for cosmetic applications. Journal of Cosmetic and Laser
Therapy 9:186–192
Carruthers J, Fagien S, Matarasso, SL, et al 2004 Consensus
recommendations on the use of botulinum toxin type A
in facial aesthetics. Plastic and Reconstructive Surgery
114:S1–S22
Chajchir I, Modi P, Chajchir A 2008 Novel topical BoNTA
(CosmeTox, Toxin Type A) cream used to treat hyperfunctional
wrinkles of the face, mouth, and neck. Aesthetic Plastic Surgery
32:715–722
Fittipaldi A, Ferrari A, Zoppé M, et al 2003 Cell membrane lipid
rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins.
Journal of Biological Chemistry 278(36):34141–34149
Glogau RG 2007 Topically applied botulinum toxin type A for
the treatment of primary axillary hyperhidrosis: results of a
randomized, blinded, vehicle-controlled study. Dermatologic
Surgery 33:S76–S80
Glogau RG 2011 In: Cohen Joel L (ed) Botulinum toxins in
dermatology: cosmetic and clinical applications. Wiley-Blackwell,
Oxford, UK.
Glogau RG, Blitzer A, Brandt F, et al 2012 Results of a randomized,
double-blind, placebo-controlled study to evaluate the efficacy
and safety of a botulinum toxin type A topical gel for the
treatment of moderate to severe lateral canthal lines. Journal of
Drugs in Dermatology 11(1):38–45
Hallett M 1999 One man’s poison–clinical applications of
botulinum toxin. New England Journal of Medicine
341:118–120
Kaplan IM, Wadia JS, Dowdy SF 2005 Cationic TAT peptide
transduction domain enters cells by macropinocytosis. Journal of
Controlled Release 102(1):247–253
likely have no effect on the combination product’s risk
profile, but would have a negative effect on the combina­
tion product’s potential benefit. The dose selected for
further clinical development is the lowest dose of each to
achieve adequate efficacy with adequate safety.
After dosage selection, efficacy of the proposed
Phase III dose of RT001 was demonstrated in two confir­
matory Phase II studies by Glogau et al. In these studies
RT001 Topical Gel demonstrated efficacy following a
30-minute application in comparison with controls for the
treatment of subjects with moderate to severe lateral
canthal lines related to orbicularis oculi muscle spasm, as
summarized in Table 10.2. End points employed both
investigators’ assessments (IGA-LCL) and subject assess­
ments (PSA) of lateral canthal line severity as well as
composites of both measures. Additionally subject self-
perception of improvement was tabulated. The 25 ng/mL
dose of RT001 demonstrated statistically significant effi­
cacy versus placebo on the primary composite endpoint as
well as all secondary endpoints and was well tolerated.
Potential applications of topical BoNT-A
Phase III clinical trials of the topical gel preparation of
RT001 are being planned now that the concentrations of
both the toxin and the peptide have been optimized.
Clinical literature and data confirm that improvement in
lateral canthal lines at rest is the most widely sought
crow’s-feet-related clinical condition, so Phase III trials
will be initiated for this indication. Earlier trials have
established the range of doses, validity, and reliability of
screening the lateral canthal lines at rest as the most stable
Table 10.2  Efficacy analyses for the proposed Phase III
dose of RT001
RT001 subjects
(25 ng)
Control
subjects
Subjects assessed at
week 4 (N)
136
132
Composite ≥2-point
improvement in PSA and
≥2-point IGA-LCL
improvement in both LCA
41.9%
p < 0.0001
0.74%
≥2-Point IGA-LCL
improvement in both LCA
51.5%
p < 0.0001
10.6%
≥2-Point improvement in
PSA
46.3%
p < 0.0001
3.0%
≥1-Point IGA-LCL
improvement in both LCA
75.7%
p < 0.0001
22.0%
PGIC improved or much
improved
52.9%
p < 0.0001
7.6%
Note: The p-value is from the Pearson’s chi-square test.

71
10 
Topical neurotoxin
Kavanagh GM, Oh C, Shams K 2004 BOTOX delivery by
iontophoresis. British Journal of Dermatology 151:1093–1095
Matarasso SL, Matarasso A 2001 Treatment guidelines for
botulinum toxin type A for the periocular region and a report
on partial upper lip ptosis following injections to the lateral
canthal rhytids. Plastic and Reconstructive Surgery 10:208–214
Schwarze SR, Hruska KA, Dowdy SF 2000 Protein transduction:
unrestricted delivery into all cells? Trends in Cell Biology
10(7):290–295
Scott AB 1981 Botulinum toxin injection of eye muscles to correct
strabismus. Transactions of the American Ophthalmological
Society 79:734–770
Solomon P 2005 Delivery of Botox by iontophoresis. British Journal
of Dermatology 153:1075
Spencer JM 2002 Botulinum toxin B. The new option in cosmetic
injection. Journal of Drugs in Dermatology 1:17–22

Reported substances used in
the reconstitution process
OnabotulinumtoxinA is provided as a vacuum-dried
powder, whereas incobotulinumtoxinA, abobotulinum­
toxinA, and BoNT-A from Lanzhou (China) are presented
as lyophilized powders (Fig. 11.1). RimabotulinumtoxinB
is provided as a ready-to-use sterile liquid and no recon­
stitution is required.
Unpreserved saline
Most manufacturers and also an international consensus
on the use of abobotulinumtoxinA recommend reconstitu­
tion of botulinum toxin A using unpreserved saline.
Preserved saline
Preserved saline contains benzyl alcohol. Three trials
including 213 patients (for cosmetic, blepharospasm, and
axillary hyperhidrosis indications) demonstrated that it
does not affect the potency of onabotulinumtoxinA and it
made injections less painful.
It was chosen by an onabotulinumtoxinA consensus
panel in 2004 as the preferred diluent for reconstitution,
and another abobotulinumtoxinA expert panel recognized
that benzyl alcohol may act as a local anesthetic, but this
effect is considered negligible at low volumes.
Hyaluronidase
Goodman tested the viability of onabotulinumtoxinA
reconstituted with saline plus hyaluronidase. Results after
2 weeks showed maintenance of efficacy and a greater
diffusion of the product. No long-term results were
evaluated.
Sterile water
BoNT-A still works if dissolved in sterile water, but intense
short-lived pain at injection site will occur.
Epinephrine and / or lidocaine
Hantash et al tested the effect of epinephrine (EPI)
1 : 100 000 on onabotulinumtoxinA efficacy in periorbital
11 
Reconstitution / Dilution
Ada R. Trindade de Almeida,
Letícia Cardoso Secco
Several situations have been implicated in affecting the
potency of BoNT. For this reason, different recommenda­
tions have been made aiming at not interfering with its
efficacy or duration. In this chapter, published information
on various methods of reconstitution, including mixtures
of toxins with several substances, agitation or foam forma­
tion, will be reviewed as well as the impact of volume on
botulinum toxin performance.
Summary and Key Features
•
Prescribing information on all available products
recommends reconstitution in unpreserved saline
•
Studies in the literature suggest that other
solutions can be used such as preserved saline,
anesthetics (lidocaine or bupivacaine), with or
without epinephrine and albumin
•
In vivo studies demonstrate that
onabotulinumtoxinA is not as fragile as originally
thought and that foam during reconstitution does
not inactivate the toxin
•
Although manufacturers recommend storage at
2–8°C in the refrigerator, toxin administration within
4–24 hours, and avoiding freezing after
reconstitution, it appears that some products can
be stored for longer periods of time after
reconstitution, in the refrigerator or the freezer,
without loss of efficacy or contamination
•
In regard to the best dilution to be used, when
treating limb muscles, perhaps as a result of
greater dispersion of the toxin, higher dilutions
might be beneficial
•
For facial muscles, more concentrated volumes are
preferred, since they produce less discomfort for
patients
•
The literature suggests that toxins may be more
sturdy and resistant to degradation than previously
understood

73
11 
Reconstitution / Dilution
procedure less painful and showed greater muscle weak­
ness at 1 week, but there were no differences at 1 and 3
months. The explanation for the faster onset of the paresis
might be a synergistic effect of bupivacaine inducing
myotoxicity.
Albumin
Bigalke et al demonstrated that human albumin supple­
mentation of abobotulinumtoxinA could allow dose
reduction without decrease of therapeutic efficacy or
safety. Their findings were confirmed by another study
with 106 patients treated with abobotulinumtoxinA, for
cervical dystonia, blepharospasm, and hemifacial spasm,
over 5–10 years.
Figure 11.1  Appearance of various vacuum-dried and lyophilized
BoNT-A powder products, before reconstitution.
Box 11.1 
Substances reported not to inactivate BoNT-A
n
Unpreserved saline
n
Preserved saline
n
Hyaluronidase
n
Distilled water
n
Epinephrine
n
Lidocaine
n
Bupivacaine
n
Albumin
n
Hyaluronic acid
Pearl 2 
Albumin supplementation of abobotulinumtoxin could allow
dose reduction without decrease of therapeutic efficacy or
safety.
Pearl 1 
The use of local anesthetics for reconstitution does not affect
botulinum toxin potency, but may increase the possibility of
an anaphylactic reaction.
rhytides in 14 patients, evaluated for up to 6 months.
They found that EPI promoted a faster onset of action
and enhanced short-term efficacy of onabotulinumtoxinA.
Gassner & Sherris, in a double-blind, randomized, con­
trolled study with 10 volunteers for cosmetic indication,
showed that when onabotulinumtoxinA was reconstituted
with 1% lidocaine with 1 : 100 000 epinephrine, all com­
ponents retained their function. Similar results were
found by Haubner in two cases, using 2% lidocaine with
1 : 200 000 epinephrine.
A cocktail composed of abobotulinumtoxinA, 2% lido­
caine with 1 : 100 000 epinephrine and hyaluronic acid
(Perlane®, Medicis, Scottsdale, AZ) was recently described.
The combined product was injected in five patients for
periocular and / or glabellar areas and the author reported
no compromise in efficacy or safety, but only periocular
post-treatment photographs were showed.
A study conducted in 29 patients, compared side by
side, onabotulinumtoxinA reconstituted in 2% lidocaine
or normal saline for axillary hyperhidrosis and found
similar effectiveness in both the short and long term. As
the lidocaine-admixed solution was associated with signifi­
cant pain reduction, it might be preferable for treating
axillary hyperhidrosis.
A fatal case of anaphylaxis after the injection of ona­
botulinumtoxinA and lidocaine mixture in a woman for
chronic neck and back pain was also reported. Since it was
not possible to determine which drug was responsible for
the reaction, it is advisable to consider that lidocaine may
increase the possibility of an anaphylactic reaction.
Bupivacaine
Yen et al, in a randomized, double-blind, split-face study
with 16 patients with glabellar wrinkles, compared ona­
botulinumtoxinA reconstituted with 0.75% bupivacaine or
with unpreserved saline on either side of their face. The
bupivacaine-reconstituted toxin treated sides made the
Box 11.1 summarizes substances reported not to inacti­
vate BoNT-A.
Variations in the reconstitution process
Foam during reconstitution
Toth et al evaluated in vitro the stability of fragments of
BoNT (light chain, LcA) and the binding domain of the
heavy chain (Hc) to mild agitation, and found that LcA
fragments rapidly lost their secondary structures and Hc
domains were denatured by mild stirring. They speculated
that BoNT domains underwent surface denaturation
owing to rapid exposure of hydrophobic residues by
mechanical agitation.
A recent experimental study evaluated the effect
of onabotulinumtoxinA after continuous inversion and
straightening of the vial, 30 times a minute, for up to 6
weeks. Eight mice were each injected intraperitoneally
with 1 U of the agitated onabotulinumtoxinA on days 1,
3, 5, 7, 14, 21, 28, and 42. The main outcome measure

74
Botulinum toxin
and injected without losing its potency for up to 6 months.
Regarding abobotulinumtoxinA, a prospective, double-
blinded, randomized controlled trial by Yang et al, with
40 subjects treated for horizontal forehead rhytides, found
no difference in efficacy after 2 weeks of refrigeration or
freezing compared with fresh product.
was death of the mice, demonstrating toxin efficacy. At
the end of the study, half of each group of mice (4 of 8
mice) died within 48 hours of the injection (range 16–48
hours), demonstrating maintenance of the onabobotu­
linumtoxinA effect.
These results confirmed previous studies in clinical set­
tings: a split-face study comparing muscle paralysis in
periocular and glabellar areas, after injection of onabotu­
linumtoxinA gently reconstituted on one side and ona­
botulinumtoxinA manually shaken on the other side,
showed no differences in short or long term effects. Kazim
et al reached similar conclusions after 6 months evaluating
frontalis muscle injections of onabotulinumtoxinA, recon­
stituted gently on one side of the face and vigorously
(placed on a vortex Touch mixer at maximum speed for
30 seconds) on the other.
Differences between in vitro and in vivo studies may
be explained by the presence of associated proteins inside
the complex of onabotulinumtoxinA, which in addition to
stabilizing the neurotoxin molecule may further protect it
from degradation.
Storage
RimabotulinumtoxinB must be stored at 2–8°C. At this
temperature, no significant loss of activity has been found
after 30 months, but at room temperature (25°C) it
dropped to 9 months.
Regarding reconstituted BoNT-A vials, manufacturers
recommend administration within 4–24 hours, storage at
2–8°C in the refrigerator, and avoidance of freezing after
reconstitution, but several publications suggest that these
recommendations may in fact be excessively strict.
Shelf life after reconstitution
A multicenter, double-blind study in 88 patients demon­
strated that reconstitution with non-preserved saline up
to 6 weeks before use did not reduce efficacy in treating
glabellar frown lines for up to 4 months, findings that were
confirmed by a recent experimental trial in mice. In the
2004 consensus panel, 73% of panelists stored onabotu­
linumtoxinA for more than 4 weeks.
For crow’s feet lines, one study used 1-week-reconsti­
tuted onabotulinumtoxinA in 30 patients, and another
used onabotulinumtoxinA after 2 weeks of refrigeration.
Both found similar clinical results to fresh product.
Another trial using 2-week-reconstituted abobotu­
linumtoxinA found no difference in safety or efficacy,
while a consensus panel in 2010 considered that it can be
used within 2–3 weeks without adverse effects.
Fresh or frozen
Parsa et al compared reconstituted onabotulinumtoxinA
frozen for up to 6 months with fresh onabotulinumtoxinA
used within 4 hours after reconstitution, in 118 sites of
80 patients; they concluded that it may be frozen, thawed,
Pearl 4 
There was no evidence of contamination in vials stored and
reused for up to 2 weeks.
Pearl 3 
Fresh versus frozen / thawed reconstituted abobotulinum­
toxinA and onabotulinumtoxinA showed no difference in
efficacy.
Sterility
Since storing and reusing reconstituted toxin for some
days or weeks has become a frequent practice, sterility
assessment is an important issue addressed by some
authors. Alam et al evaluated 127 vials of onabotulinum­
toxinA that were reconstituted with preservative-
containing saline, stored and reused, simulating routine
clinical practice. Each vial underwent an average of 4.5
access procedures during a period of up to 7 weeks, and
there was no evidence found of contamination in sterility
analysis. Menon analyzed 11 consecutive bottles of abobo­
tulinum toxinA left for 4 hours at room temperature and
found no microbial growth; there were similar findings in
8 vials of this product kept refrigerated for 15 days after
reconstitution.
Dilution issues
There is considerable discussion about the effect of dilu­
tion of BoNTs on clinical effect, duration and diffusion.
Table 11.1 shows the BoNT-A manufacturers’ recom­
mended volumes for reconstitution.
Rimabotulinumtoxin B can be diluted up to sixfold
using normal saline, but a more concentrated solution
cannot be generated.
Some researchers believe that higher dilutions could be
associated with a higher risk of diffusion to unwanted
Table 11.1  BoNT-A manufacturers’ dilution
recommendations
Toxin
Dilution range
Concentration
OnabotulinumtoxinA
1–8 mL of saline
12.5–100 U/mL
AbobotulinumtoxinA
300 U in
0.6–2.5 mL
120–500 U/mL
IncobotulinumtoxinA
1–8 mL
12.5–100 U/mL

75
11 
Reconstitution / Dilution
dilutions was reported with respect to either efficacy or
complications.
Non-facial muscles
Experimental studies
Shaari et al treated the tibialis anterior muscle in rats and
found that a constant dose of 0.2 U of botulinum toxin
resulted in greater paralysis when injected in larger
volumes. Kim et al injected 16 rabbits with 10 U of ona­
botulinumtoxinA at concentrations of 20 or 100 U/mL
and drew the same conclusions.
Limb muscle dystonias / spasticity
A group from Korea injected a fixed dose of 2.5 U of
onabotulinumtoxinA to human extensor digitorum brevis
muscle in two dilutions (5 and 25 U/mL) and concluded
that a fivefold increase in dilution volume did not enhance
the paralyzing effect of onabotulinumtoxinA. Francisco
et al treated 13 patients with 60 U diluted to 50 or
100 U/mL for wrist and finger flexor spasticity and also
found no significant differences.
In contrast, Gracies et al used 160 U of onabotulinum­
toxinA at two different dilutions (20 and 100 U/mL), in
a double-blind randomized controlled trial with 21 indi­
viduals with spastic elbow flexors. They found that the
higher-volume group (20 U/mL) achieved greater neuro­
muscular blockade and spasticity reduction, compared
with the concentrated solution.
Two studies were conducted in children with cerebral
palsy and limb spasticity. One used onabotulinumtoxinA
in 38 children at concentrations of 12.5 (n = 19) and
50 U/mL (n = 19) respectively in the gastrocnemius
muscles for reducing ankle plantar flexor spasticity.
Although no significant differences were found in physical
evaluation or gait analysis, the larger-volume group had
more frequent side effects, making the more concentrated
solution a better option. In another randomized controlled
trial, 22 children with cerebral palsy received a fixed dose
of abobotulinumtoxinA. Dilutions of 500 U/5 mL and
500 U/1 mL respectively were injected in each gastroc­
nemius muscle. The authors considered the high-volume
preparation more effective.
sites, unsatisfactory results or shorter duration, but this is
not unanimous in the literature. To facilitate the under­
standing of the dilution impact on diffusion, adverse
effects and success, the available studies were reviewed
by the authors in this regard.
Facial muscles
Cosmetic indications
Expert consensus panels for cosmetic indications re­
commend dilutions between 1 and 3 mL of saline for
onabotulinumtoxinA and between 1.5 and 2.5 mL for
abobotulinumtoxinA.
In the past, a group reported the use of large volumes
of low-dose BoNT-A (up to 20 mL/vial) and described
efficacy, but of short duration. The need for frequent
treatments to maintain results led to a great number of
individuals resistant to toxin. Therefore, higher dilutions
are no longer recommended.
Hankins et al evaluated 46 patients with glabellar wrin­
kles injected with different concentrations of onabotu­
linumtoxinA (10 or 100 U/mL) for up to 21 weeks and
found no differences in efficacy or duration, but larger
volumes were associated with more discomfort.
In a study with 10 volunteers, Hsu et al injected the
forehead with 5 U of onabotulinumtoxinA diluted to 20
(5 mL) or 100 U/mL (1 mL). They found that higher-
volume injections resulted in greater diffusion and a larger
affected area. Carruthers et al studied the effect of dif­
ferent dilutions of onabotulinumtoxinA in the treatment
of glabellar rhytides. Eighty patients were treated with
dilutions of 10, 20, 33.3, or 100 U/mL (20/group) and
followed for 48 weeks. No statistical differences in treat­
ment success or adverse effect were reported, although
the six cases of eyebrow ptosis were all in the higher-
dilution group.
Another trial treated 20 patients for lateral orbital rhyt­
ides, observed over 90 days. Dilutions of onabotulinum­
toxinA were 20 or 100 U/mL. No statistically significant
differences in response or adverse effects were detected,
even though the lower-dilution group showed a slightly
better response.
For incobotulinumtoxinA, two different dilutions (25
and 40 U/mL) were evaluated in a split-face study in 40
patients with glabellar lines; it found no significant differ­
ences between sides.
Pearl 6 
When treating limb muscles, higher dilutions might be
beneficial, perhaps due to a greater dispersion of the toxin.
Pearl 5 
For cosmetic indications (i.e. facial muscles), concentrated
solutions seem better than diluted ones because they
produce less discomfort for patients.
Blepharospasm
Sixteen benign essential blepharospasm patients were
treated with concentrations of 10 or 100 U/mL, and fol­
lowed for 8 months. No significant difference between
Hyperhidrosis
There is no standardized dilution for botulinum toxin
treatment of focal hyperhidrosis. Dilutions reported for
hyperhidrosis are detailed in Table 11.2.
Open comparative trials in hyperhidrotic areas assessed
by iodine starch tests were not conclusive: a study of 9

76
Botulinum toxin
Boyle MH, McGwin, Flanagan CE, et al 2009 High versus low
concentration botulinum toxin A for benign essential
blepharospasm: does dilution make a difference? Ophthalmic
Plastic and Reconstructive Surgery 25:81–84
Carruthers J, Carruthers A 2009 Botulinumtoxin in facial
rejuvenation: an update. Dermatologic Clinics 27:417–425
Carruthers A, Carruthers J, Cohen J 2007 Dilution volume of
botulinum toxin type a for the treatment of glabellar rhytides:
does it matter? Dermatologic Surgery 33:S97–S104
Carruthers J, Fagien S, Matarasso SL 2004 Consensus
recommendations on the use of botulinum toxin Type A in facial
aesthetics. Plastic and Reconstructive Surgery 114(6 suppl):
1S–22S
Gracies JM, Lugassy M, Weisz DJ, et al 2009 Botulinum toxin
dilution and endplate targeting in spasticity: a double-blind
controlled study. Archives of Physical Medicine and
Rehabilitation 90:9–16
Hexsel DM, Trindade de Almeida AR, Rutowithsch M, et al 2003
Multicenter, double blind study of the efficacy of injections
with botulinum toxin type A reconstituted up to six consecutive
weeks before application. Dermatologic Surgery 29:523
Hu GC, Chuang YC, Liu JP, et al 2009 Botulinum toxin (Dysport)
treatment of the spastic gastrocnemius muscle in children with
cerebral palsy: a randomized trial comparing two injection
volumes. Clinical Rehabilitation 23(1):64–71
Kane M, Donofrio L, Ascher B, et al 2010 Expanding the use of
neurotoxins in facial aesthetics: A consensus panel’s assessment
and recommendations. Journal of Drugs in Dermatology
9(1):S3–S22
Kazim NA, Black EH 2008 Botox: shaken, not stirred. Ophthalmic
Plastic and Reconstructive Surgery 24(1):10–12
Lee LR, Chuang YC, Yang BJ, et al 2004 Botulinum toxin for lower
limb spasticity in children with cerebral palsy: a single-blinded
trial comparing dilution techniques. American Journal of
Physical Medicine and Rehabilitation 83:766–773
Mohammadi B, Kollewe K, Wegener M, et al 2009 Experience with
long-term treatment with albumin-supplemented botulinum
toxin type A. Journal of Neural Transmission 116:437–441
Parsa AA, et al 2007 Reconstituted botulinum type A neurotoxin:
clinical efficacy after long-term freezing before use. Aesthetic
Plastic Surgery 31:188–191
Shome D, Nair AG, Kapoor R, et al 2010 Botulinum toxin A: is it
really a fragile molecule? Dermatologic Surgery 36:2106–2110
Toth SI, Smith LA, Ahmed SA 2009 Extreme sensitivity of
botulinum neurotoxin domains towards mild agitation. Journal
of Pharmaceutical Sciences 98(9):3302–3311
Trindade de Almeida AR, Kadunc BV, Di Chiacchio N, et al 2003
Foam during reconstitution does not affect the potency of
botulinum toxin A. Dermatologic Surgery 29:530
Trindade de Almeida AR, Secco LC, Carruthers A 2011 Handling
botulinum toxins: an update literature review. Dermatologic
Surgery 37(11):1553–1565
volunteers injected in the axillary region with 3 U of
onabotuliumtoxinA diluted in 1–5 mL of saline solution
reported no difference in anhydrotic halos; in addition 3
patients with compensatory hyperhidrosis received injec­
tions of 5 U of abobotulinumtoxinA in the back, in three
dilutions of 0.2, 0.4, or 0.6 mL. Although larger anhidrotic
halos were found with the more diluted abobotulinum­
toxinA group, the authors considered the differences to
be irrelevant.
We reviewed 13 comparative dilution studies: five of
these reported a greater diffusion or enhanced effect with
higher volumes, whereas nine found no difference in effi­
cacy. Three trials reported more discomfort with higher
dilutions, even though these were not statistically signifi­
cant. Hence this topic remains controversial. For small
muscles, such as those located on the face or hand, it
seems that there is no difference in results with the addi­
tion of higher volumes. For large muscles of the limbs,
though, this might be advantageous. More studies, with
larger samples, are needed to clarify this matter.
Many of the precautions surrounding BoNT use, often
recommended by the manufacturers, are described in the
clinical literature as very restrictive. The current literature
suggests rather that toxins may be more sturdy and resis­
tant to degradation than was previously understood.
Further reading
Asher B, Talarico S, Casuto D, et al 2010 International consensus
recommendations on the aesthetic usage of botulinum toxin
type A (Speywood Unit) – part I: upper facial wrinkles. Journal
of the European Academy of Dermatology and Venereology
24(11):1285–1295
Table 11.2  Dilutions reported for hyperhidrosis
Toxin
Dilution range
Most used
dilution
OnabotulinumtoxinA
1–10 mL of saline
2–5 mL
AbobotulinumtoxinA
1.25–10 mL
2.5–5 mL
IncobotulinumtoxinA
1–10 mL of saline
10 mL
(one paper)

Introduction
Botulinum toxin type A has been found to be useful for
many applications since its initial introduction, but one of
its most popular uses has been for treatment of glabellar
rhytides. In 1979, the Food and Drug Administration (FDA)
gave limited approval for trials of botulinum toxin A for
strabismus. In 1985 it was used for blepharospasm. Two
years later, in 1987, Dr Jean Carruthers made an observa­
tion that patients who received botulinum toxin treatment
for blepharospasm also had improvement of dynamic gla­
bellar rhytides. The first published reports of the use of
botulinum toxin for the treatment of facial lines were those
by Carruthers & Carruthers and by Borodic in the early
1990s. However, it wasn’t until 2002 that botulinum toxin
type A was granted its first approval for cosmetic use specifi­
cally for the treatment of glabellar rhytides.
The use of botulinum toxin type A over the last two
decades for treatment of glabellar rhytides has revolution­
ized the field of cosmetic dermatology and plastic surgery.
Several multicenter, double-blind, randomized, placebo-
controlled studies have proven its efficacy (e.g. that by
Carruthers et al in 2003). It is the first quick, less invasive
and effective, non-surgical technique for both brow lift
and rhytid treatment. The same group demonstrated in
2010 that botulinum toxin type A injection to the glabella
has been shown to improve both dynamic glabellar rhyt­
ides and those in repose. Subsequent uses of botulinum
toxin type A for cosmetic improvement of forehead, peri­
oral area, chin, crow’s feet, and other rhytides have been
successful, but its use in the glabella is the first and still
the most widely used.
In this chapter, we will discuss the anatomy, injection
technique, dosing, special considerations, and potential
adverse events of botulinum toxin injection of the
glabella.
Anatomy
The glabellar complex consists of the two corrugator
supercilii muscles and the procerus muscle that collec­
tively serve upon contraction to pull the brow medially
and downward (Fig. 12.1). The corrugator supercilii are
two sets of horizontally oriented muscle fibers that lie
beneath the medial eyebrow to about the mid-pupillary
line. In some patients, the corrugators extend beyond the
mid-pupillary line (these muscles can be visualized at
maximum contraction when ‘frowning’). The procerus is
a vertically oriented muscle that lies in between the eye­
brows. The frontalis muscle of the forehead is vertically
oriented and the medial belly interpolates with the glabel­
lar complex, and its lateral portion interpolates with the
lateral orbicularis oculi. Its main function is to elevate the
brow. The orbicularis oculi is a thin circular muscle around
the eyes that lies on top of the lateral portion of the cor­
rugator supercilli. The lateral portion of the orbicularis
oculi under the tail of the brow is a powerful brow depres­
sor upon contraction. The levator palpebrae muscle lies
beneath the orbicularis oculi, underneath the bony orbital
rim, and its function is eyelid opening. Rhytides are typi­
cally perpendicular to the orientation of muscle fibers,
thus contraction of the glabellar muscles typically pro­
duces vertically oriented lines between the brows.
Injection technique (see Video ‘Botulinum
Toxin Glabella’)
The proper preparation, storage, and handling of bot­
ulinum toxin is discussed in Chapters 9 and 11. Insulin
12 
Glabella
Naissan O. Wesley, Derek H. Jones
Summary and Key Features
•
Botulinum toxin type A for treatment of glabellar
rhytides was FDA approved for cosmetic use in
2002, but has been used for this purpose for over
20 years
•
Injection of botulinum toxin into the glabellar
complex (corrugator supercilii and procerus
muscles) and the lateral eyebrow may achieve
reduction of glabellar rhytides and lower forehead
rhytides, and brow lift
•
There are currently three types of botulinum type A
toxin injectables specifically FDA approved for the
treatment of glabellar furrows, including Botox®,
Dysport®, and Xeomin®, at doses of 20 units (U),
50 U, and 20 U respectively
•
Dose variation may be required to obtain optimal
correction and higher doses may be required in
men

78
Botulinum toxin
patients with particularly long or stronger corrugator
muscles, an additional injection site may be given midway
between the medial and lateral corrugator injection sites
(a total of seven injections).
syringes or 1 mL syringes with 30–32 gauge needles are
typically used for injection.
In the glabella, there are typically five injection sites:
one at each medial corrugator, one at each lateral corruga­
tor (1 cm above orbital rim at the mid-pupillary line), and
a single injection into the procerus (Fig. 12.2). For some
Figure 12.1  Muscles of the face. Courtesy Dr Jean Carruthers,
reproduced with permission.
frontalis
procerus
corrugator
supercilii
obicularis
oculi
Figure 12.2  Five injection sites for botulinum toxin into the glabella:
one at each medial corrugator, one at each lateral corrugator (1 cm
above the orbital rim at the mid-pupillary line), and a single injection
into the procerus.
Pearl 3 
To achieve ‘brow lift’, an additional injection of 3 U may be
given at each lateral brow (directly under the hair-bearing tail
of the brow, to relax the depressor portion of orbicularis
oculi). As the orbicularis oculi is a circular muscle, the superior
lateral portion acts as a depressor of the brow when
contracted, thus relaxation of this muscle (in addition to
relaxation of the depressor muscles of the glabellar complex)
results in a ‘brow lift’.
Pearl 2 
Injection of botulinum toxin in to the corrugators supercilii and
procerus not only reduces glabellar rhytides but also reduce
those of the lower half of the forehead by diffusion to the
lower half of the medial belly of the frontalis muscle.
Pearl 1 
While directly facing the patient, ask him or her first to stare
at a midline point on your face and then to frown, in order
for you to visualize the corrugator supercilii and procerus
muscles in contraction, and to visualize the mid-pupillary line
with exact symmetry. Some may consider marking the
injection sites with a marker or lip liner pencil on the patient’s
skin prior to actual injection. For precision, rest the thumb of
the non-injecting hand on the superior orbital rim, and have
the patient contract while holding the corrugator between the
thumb and forefinger. This allows for definitive isolation of
the muscle, and prevents accidental injection below the
infraorbital rim, which may increase the risk of lid ptosis. In
addition, one may steady the injecting hand by resting the
lateral fingers or hand on the patient’s nose or cheek.
Techniques for decreasing pain include intrafollicular orifice
injection, applying ice or topical anesthetic to the skin pre-
injection, using a smaller gauge needle (30-, 31-, or 32-gauge
needle), using preservative-containing isotonic sodium chlo­
ride solution for dilution, and ensuring that patients are not
contracting their muscles during actual injection.
Even without the lateral brow injection, injection of
20–40 units of botulinum toxin type A into the glabellar
muscles alone leads to eyebrow elevation. This is due to
relaxation of the depressor actions of the corrugator pro­
cerus muscles, as well as inactivation of the medial muscles
of the frontalis, with resultant increased muscle tone of
the lateral and superior muscles of the frontalis. Studies
by Huilgol et al and by Huang and co-workers found that
the eyebrow elevation usually ranges from 1 to 3 mm
(mean elevation 1 mm).
Dosing
Botox® (onabotulinumtoxinA) (Fig. 12.3)
With standard dilutions of a 100-unit vial of Botox® (Aller­
gan Inc., Irvine, CA, USA) with 2.5 mL of 0.9% normal
saline, a single injection of 0.1 mL yields 4 units. In
women, 20–24 units of Botox® via five injection points are
typically injected into the glabellar complex; an additional

79
12 
Glabella
in 523 subjects of diverse ethnic backgrounds, treatment
with a 20 U dose resulted in more than half of the
responders sustaining clinical benefit for 4 months. In fact,
Dailey and co-workers found that injection of 20 U of
onabotulinumtoxinA to the glabellar complex every 4
months for 20 months significantly reduces or progres­
sively eliminates glabellar rhytides for up to 6 months
after the last treatment.
Dysport® (abobotulinumtoxinA)
(Figs 12.4 and 12.5)
Dysport® (Ipsen Biopharm Ltd, Wrexham, England,
distributed by Medicis Pharmaceuticals, Scottsdale,
AZ, USA) is a clostridium botulinum type A toxin–
hemagglutin complex. In a randomized, double-blind,
placebo-controlled study of Dysport® by Monheit et al to
determine the optimal dose for treatment of glabellar
3 units may be injected into each lateral eyebrow to
achieve brow lift. In our experience, in men, 20–40 or
more units may be injected into the glabellar complex
depending on the strength of muscle contraction. In a 2005
study by Carruthers and colleagues of 80 men randomized
to receive 20, 40, 60, or 80 units of botulinum toxin type
A (Botox® or Vistabel®, Allergan Inc., Irvine, CA), the 40,
60, and 80 U doses were consistently more effective than
the 20-unit dose in reducing glabellar lines (duration, peak
response rate, improvement from baseline).
Onset of response is typically 1–14 days and results last
for 3–4 months. In a 2011 study by Beer et al of 45
patients who received a 20 U injection into the glabella,
nearly half of patients may experience onset of response
by day 1, with 100% by day 14. A meta-analysis by Kane
and colleagues of the duration of efficacy pooled results
from four global Phase III pivotal trials of onabotulinum­
toxinA treatment of glabellar lines and demonstrated that,
Figure 12.3  (A) Pre-onabotulinumtoxinA injection, relaxed. (B) Pre-onabotulinumtoxinA in maximal glabellar contraction. (C) Pre-
onabotulinumtoxinA in maximal frontalis contraction. (D) 1 week after 20 U onabotulinumtoxinA injection to the glabella, relaxed. (E) 1 week
after 20 U onabotulinumtoxinA injection to the glabella, in maximal glabellar contraction. (F) 1 week after 20 U onabotulinumtoxinA injection to
the glabella, in maximal frontalis contraction.
A
B
C
F
E
D

80
Botulinum toxin
studies by Kranz et al and by Carli et al, in mice and
human skin models respectively, suggest that at equal unit
doses (i.e. 20 U Botox® compared with 20 U Dysport®),
there is no difference in diffusion characteristics or spread.
Spread is likely to increase as the unit dose increases. As
a larger number of units of Dysport® are required for a
similar clinical effect compared with Botox®, the spread
of effect may be larger. The onset of action of Dysport®
may be 1–2 days earlier, but Nestor et al found both
products to have similar efficacy.
Xeomin® (incobotulinumtoxinA) (Fig. 12.6)
Xeomin® (Merz Pharmaceuticals, Frankfurt, Germany) is
a recently approved botulinum toxin type A that is report­
edly devoid of the complexing proteins that naturally
encapsulate the 150 kDa active portion of the botulinum
toxin A molecule. Xeomin® is available in 50 and 100 U
per vial, the same as Botox®. Studies by Dressler and col­
leagues have shown that the potency of Xeomin® and
Botox® is equivalent, and that conversion of doses may be
performed in a 1 : 1 ratio; when five unexpired batches of
onabotulinumtoxinA (Botox®) and incobotulinumtoxinA
(Xeomin®) were compared in batch release assay, statisti­
cal analyses failed to detect differences in the potency
labeling of the two products. Sattler et al demonstrated
lines, participants were given 20, 50, or 75 U of Dysport®
or placebo, and followed up on days 7, 30, 60, 90, and
120. Dysport® treatment resulted in a significant improve­
ment in glabellar lines compared with placebo; the 50 U
dose was determined to be optimal. Flynn found that, like
Botox®, glabellar retreatment intervals are every 3–4
months (with a mean of 3.9 months, median 3.3 months).
A single comparative study of Botox® and Dysport® by
Lowe and colleagues demonstrated that the proportion of
patients relapsing at week 16 was 23% (95% CI 11.5,
41.6) in the Botox® group, as compared with 40% (95%
CI 25.2, 60.1) in the Dysport® group.
For treatment of glabellar lines, Dysport® is packaged
in a 300 U bottle. Manufacturer recommendations for
reconstitution are with 2.5 mL or 1.5 mL of 0.9% sodium
chloride injection USP (without preservative), to yield a
solution of 10 U per 0.08 mL with 2.5 mL, or a solution
of 10 U per 0.05 mL with 1.5 mL. The manufacturer also
recommends 50 U given intramuscularly in five equal ali­
quots of 10 U each are recommended to achieve clinical
effect.
Two clinical studies (by Trindade et al and Roche et al)
have suggested that there may be a greater spread or dif­
fusion (the so-called ‘action halo’) of Dysport® from the
site of injection compared with Botox®, especially when
dosed appropriately for efficacy. However, two other
Figure 12.4  Maximal glabellar contraction: (A) pre-injection and (B) 14 days after 50 U injection of abobotulinumtoxinA in to the glabella,
female patient. Reproduced with permission of Medicis Pharmaceutical Corporation. ©2012 Medicis Pharmaceutical Corporation. All rights
reserved.
A
B
Figure 12.5  Maximal glabellar contraction: (A) before and (B) 14 days after 50 U injection of abobotulinumtoxinA in to the glabella, male
patient. Reproduced with permission of Medicis Pharmaceutical Corporation. ©2012 Medicis Pharmaceutical Corporation. All rights reserved.
A
B

81
12 
Glabella
Figure 12.6  IncobotulinumtoxinA (A) Before: at rest and (B) during contraction. (C) After: at rest and (D) during contraction. Reproduced with
permission of Medicis Pharmaceutical Corporation. ©2012 Medicis Pharmaceutical Corporation. All rights reserved.
B
A
C
D