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CURRENT OPINION
Pharmaceutical, Biological, and Clinical Properties of Botulinum
Neurotoxin Type A Products
Ju
¨rgen Frevert
Published online: 6 January 2015
ÓThe Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Botulinum neurotoxin injections are a valuable
treatment modality for many therapeutic indications and
have revolutionized the field of aesthetic medicine so that
they are the leading cosmetic procedure performed
worldwide. Studies show that onabotulinumtoxinA, abob-
otulinumtoxinA, and incobotulinumtoxinA are comparable
in terms of clinical efficacy. Differences between the pro-
ducts relate to the botulinum neurotoxin complexes, spe-
cific biological potency, and their immunogenicity. Protein
complex size and molecular weight have no effect on
biological activity, stability, distribution, or side effect
profile. Complexing proteins and inactive toxin (toxoid)
content increase the risk of neutralizing antibody forma-
tion, which can cause secondary treatment failure, partic-
ularly in chronic disorders that require frequent injections
and long-term treatment. These attributes could lead to
differences in therapeutic outcomes, and, given the wide-
spread aesthetic use of these three neurotoxin products,
physicians should be aware of how they differ to ensure
their safe and effective use.
1 Introduction
Botulinum toxin is produced by anaerobic fermentation of
the bacterium Clostridium botulinum. A number of differ-
ent strains of C. botulinum have been identified, which
produce eight immunologically distinct serotypes (type A–
H) and consist of the botulinum neurotoxin complexed
with a number of neurotoxin-associated proteins. Serotypes
A and B have been developed for human use. The first
approval of a botulinum neurotoxin was for the treatment
of blepharospasm, hemifacial spasm, and strabismus in
1989. Since then, the number of commercial botulinum
toxins and their uses has expanded for clinical as well as
aesthetic indications, and botulinum toxin products are now
licensed for a broad range of indications (with approved
indications varying by country and product), including
temporary improvement of dynamic facial lines, symp-
tomatic relief of blepharospasm, cervical dystonia (spas-
modic torticollis), and various forms of focal spasticity,
management of severe hyperhidrosis, prophylaxis of
headaches in adults with chronic migraine, and manage-
ment of urinary incontinence due to spinal cord injury or
multiple sclerosis [1–6].
Neurotoxin type A preparations are the most widely
used worldwide and the only ones that are US FDA
approved for aesthetic use. There are currently three
leading botulinum neurotoxin type A (BoNT/A) products
on the market in the Western hemisphere: onabotulinum-
toxin A (ONA; Botox/Vistabel
Ò
, Allergan Inc., Irvine, CA,
USA), abobotulinumtoxin A (ABO; Dysport
Ò
/Azzalure
Ò
,
Ipsen, Paris, France), and incobotulinumtoxin A (INCO;
Xeomin/Bocouture
Ò
, Merz Pharmaceuticals GmbH,
Frankfurt, Germany). Recently published statistics from the
International Society of Aesthetic Plastic Surgery show
that BoNT/A injections are now the most popular of all
cosmetic procedures worldwide, both surgical and non-
surgical [7]. As a result of the growing popularity of BoNT/
A injections among the general public, physicians from
diverse specialties are integrating botulinum toxin injec-
tions into their practices. With three BoNT/A products now
available for aesthetic use and a large population of
potential patients with individual needs and preferences, it
is important that practitioners are familiar with all aspects
J. Frevert (&)
Head of Botulinum Toxin Research, Merz Pharmaceuticals
GmbH, Hermannswerder 15, 14473 Potsdam, Germany
e-mail: juergen.Frevert@merz.de
Drugs R D (2015) 15:1–9
DOI 10.1007/s40268-014-0077-1
of the available preparations. The author of this paper has
been involved in botulinum neurotoxin research since 1983
and was instrumental in the development of the latest
BoNT/A product to reach the market. In this current
opinion paper, the author highlights the similarities and
differences between the currently available BoNT/A pro-
ducts in terms of their pharmaceutical and biological
properties, and discusses why an understanding of these
properties is important for optimal therapeutic use.
2 Clinical Comparisons
Several clinical studies in different indications (including,
but not limited to, cervical dystonia, glabellar lines, crow’s
feet, and blepharospasm) have demonstrated comparable
clinical efficacy of INCO compared with ONA, with a 1:1
conversion ratio between the two products [8–12]. The
studies also reported very similar outcomes in terms of
time to onset, time to waning, total duration of effect, and
side effect profile.
Some publications give approximate conversion factors
between preparations, with several reporting that the units
of ONA and INCO are equivalent [13–17]. Recent evi-
dence-based consensus reviews on BoNT/A applications in
aesthetic medicine have summarized the evidence sup-
porting a 1:1 dose relationship between ONA and INCO
[18–20]. When converting between ABO and ONA, a ratio
of 2–3 ABO units for 1 unit of ONA has been proposed in
several publications [17,21,22]. A consensus review by
Carruthers et al. [18] stated that, although no clear
ONA:ABO conversion ratio has been established, a dose
ratio of 1:2.5 may be assumed in aesthetic indications.
3 Structure and Mechanism of Action
Botulinum toxin consists of the 150 kDa neurotoxin itself
and a set of neurotoxin-associated complexing proteins
(NAPs), which together form high-molecular-weight pro-
genitor complexes. All botulinum neurotoxin serotypes are
synthesized as single chain proteins (150 kDa) that are
proteolytically cleaved into di-chain proteins consisting of
a 50 kDa light chain and a 100 kDa heavy chain, connected
by a disulfide bond.
All three preparations of BoNT/A have a similar
mechanism of action [23,24]. The heavy chain binds to
specific glycolipids, gangliosides (GT1b), and a specific
cell surface receptor (SV2) on cholinergic nerve endings,
enabling its uptake by endocytosis and promoting translo-
cation of the light chain across the endosomal membrane
and into the cytosolic compartment [24]. At the same time,
the disulfide bond linking the two chains is reduced,
allowing the light chain to diffuse freely in the cytosol. The
light chain has proteolytic activity and, after internaliza-
tion, binds with high specificity to a SNARE protein, which
is subsequently cleaved. The target SNARE proteins vary
among the different serotypes, but the BoNT/A serotype
cleaves synaptosomal membrane-associated protein
25 kDa (SNAP-25). The cleavage of SNAP-25 prevents the
fusion of the synaptic vesicle with the presynaptic mem-
brane, thereby blocking the release of acetylcholine into
the synaptic cleft [25,26]. Depending on the target tissue,
BoNT/A can block the cholinergic neuromuscular inner-
vation of striated and smooth muscles or the cholinergic
autonomic innervation of exocrine glands.
4 Molecular Weight and Complexing Proteins
Commercially available BoNT/A formulations contain
different complements of NAPs, and therefore have dif-
ferent molecular weights and three-dimensional structures
(Table 1)[27,28]. Studies have shown that the complex
composition of botulinum neurotoxins is specific to the
method of growth and the method of purification [29,30].
The complexes dissociate almost instantaneously on
reconstitution of the lyophilized or vacuum-dried product
[31] and have never been demonstrated in the vials of the
commercial products.
The active protein in all commercially available pro-
ducts is the 150 kDa neurotoxin, the amino acid sequence
of which is identical in ONA and INCO as they are both
produced from the Hall strain for C. Botulinum type A [32].
The corresponding sequence for ABO has not been pub-
lished, but is likely identical, because the manufacturer also
uses a Hall strain [29].
INCO differs from ONA and ABO in that it is free from
complexing proteins and consists of only the 150 kDa
neurotoxin responsible for the therapeutic effect [32].
NAPs are not pharmacologically active on nerve terminals
and consist of several hemagglutinins (HA) and a single
non-toxic non-hemagglutinin (NTNH) [27,33,34]. Data
support the role of these proteins in protecting the neuro-
toxin from acidic and proteolytic degradation in the
digestive tract [35,36]. The HAs play a key role in the oral
toxicity of botulinum neurotoxin. By binding and disrupt-
ing the cell adhesion protein E-cadherin, they allow the
toxin to pass through the intestinal epithelial barrier and
enter the systemic circulation [37].
The botulinum neurotoxin-NAP progenitor complexes
isolated from C. botulinum type A cultures adopt three
sizes: 900, 500, and 300 kDa [38]. The complex size for
ONA is 900 kDa [30] (Table 1). There is no information on
the exact complex size of ABO, but data have shown that
ABO complexing proteins are present as both full-length
2 J. Frevert
proteins and as a succession of fragments [29]. As most of
the NTNH is truncated in ABO, one can infer that there is
little or no 500 kD and no 900 kDa complex, and that the
300 kDa complex is probably the most abundant.
Importantly, it has been argued that molecular weight or
protein complex size do not affect biological activity and
pharmacological properties, as the BoNT/A neurotoxin
rapidly dissociates from the complexing proteins (if pres-
ent) after dilution, drying, and reconstitution of the prep-
aration, with C85 % of neurotoxin present in the 150 kDa
free form prior to injection into target tissues [31].
5 Botulinum Neurotoxin Type A Manufacturing
Process and Reconstitution
The precise details of manufacturing processes are pro-
prietary, but product purification involves precipitation as
a first step for each commercially available preparation.
ONA is purified by repeated precipitation and redissolu-
tion, whereas ABO is produced by purification using
column chromatography [39]. During the manufacture of
INCO, the complexing proteins are removed. This is
performed in a series of chromatographic steps to mini-
mize the risk of inactive toxin content, and thus limit
possible denaturation, degradation, and loss of biological
activity [40]. The three commercial preparations of
BoNT/A discussed in this paper are supplied in either
vacuum-dried or lyophilized (freeze-dried) form
(Table 1). Excipients (NaCl or sucrose or lactose, and
human serum albumin) are added to minimize the risk of
product inactivation during this process and during long-
term storage. ONA is diluted in a solution containing
NaCl and albumin prior to vacuum-drying, which has
been proposed to negatively impact on neurotoxin activity
and may be responsible for its toxoid (inactive neuro-
toxin) content [32,41]. From a clinical perspective, the
implication is that inactive neurotoxin would not be taken
up by nerve cells, but could be recognized by the immune
system.
Practitioners should be aware that suboptimal reconsti-
tution of BoNT/A preparations can diminish their efficacy
[42,43]. The complexing proteins dissociate almost com-
pletely from the neurotoxin following reconstitution with
saline before injection into the target. The pH of the saline
used for their reconstitution has been reported to vary
between pH 4.5 and 7.0 [44], which provides a slightly
acidic solution because the products are not buffered. A
low pH can cause a stinging sensation reported by sensitive
patients, but this is true for all products.
Table 1 Comparison of botulinum neurotoxin type A formulations
Botulinum toxin type A ABO ONA INCO
Brand name Azzalure
Ò
, Dysport
Ò
Botox
Ò
, Vistabel
Ò
Xeomin
Ò
, Bocouture
Ò
Approved aesthetic indication Moderate to severe glabellar lines Moderate to severe glabellar lines
and crow’s feet
Moderate to severe glabellar
lines and crow’s feet
Presentation Freeze-dried (lyophilized) powder
for reconstitution
Vacuum-dried powder for
reconstitution
Freeze-dried (lyophilized)
powder for reconstitution
Isolation process Precipitation and chromatography Precipitation Precipitation and
chromatography
Composition Clostridium botulinum toxin type
A; HA and non-HA proteins
Clostridium botulinum toxin type
A; HA and non-HA proteins
Clostridium botulinum toxin
type A
Excipients
a
500 U vial: human serum albumin
125 lg; lactose 2.5 mg
100 U vial: human serum albumin
0.5 mg; NaCl 0.9 mg
100 U vial: human serum
albumin 1 mg; sucrose 4.6 mg
Molecular weight (neurotoxin),
kDa
Not published (150) 900 (150) 150
Approximate total clostridial
protein content (ng per 100 U)
4.87 5.0 0.44
Neurotoxin protein load (ng
neurotoxin per 100 U
a
)
0.65 0.73 0.44
Specific neurotoxin potency (U/
ng)
154 137 227
Shelf-life 2–8 °C 2 years 2–8 °C 2–3 years
b
(or freezer) Room temperature 3–4 years
b
Storage (post-reconstitution) 2–8 °C 4 h 2–8 °C 24 h 2–8 °C24h
ABO abobotulinumtoxin A, HA hemagglutinin, INCO incobotulinumtoxin A, ONA onabotulinumtoxin A
a
Units of measurement for the three commercially available BoNT/A preparations are proprietary to each manufacturer and are not
interchangeable
b
Depending on the number of units
Pharmaceutical, Biological, and Clinical Properties of BoNT/A Products 3
6 Potency per Unit Weight of Toxin Protein
For safety and efficacy reasons, it is important for BoNT/A
biological activity to be accurately determined. The bio-
logical potency of BoNT/A drugs is based on the deter-
mination of the median lethal dose of toxin/neurotoxin
after intraperitoneal injection in mice (median lethal dose
[LD50] assay) [3,6]. On this basis, 1 unit of toxin is
defined as one mouse LD50, i.e. the dose of toxin/neuro-
toxin capable of killing 50 % of a group of mice. Product
dose for treating patients is determined by each manufac-
turer’s LD50 potency assay results [45]. These assays use
different in-house diluents and standards, so the unit of
measurement for the three commercially available BoNT/A
preparations is proprietary to each manufacturer [21]. This
precludes direct comparisons of potency between products
[29,46–48] and highlights the importance of clinical head-
to-head studies for comparing different BoNT/A products
and their respective conversion ratios. Nevertheless,
labeled potency for ONA and INCO is identical, with a 1:1
conversion ratio between the products [6,46], and several
studies have reported clinical equipotency for these agents
[8–10]. Measurement of ONA and INCO in the same LD50
assay using diluent as in clinical setting conditions showed
equivalent potency [13]. As required by governmental
agencies, the LD50 assay is being replaced by more
humane, cell-based assays, which must be cross-validated
against the LD50 assay to provide the same potency result.
Each manufacturer is developing their own proprietary
cell-based assay.
The respective amounts of neurotoxin per 100 U, mea-
sured using a high-sensitivity enzyme-linked immunosor-
bent assay (ELISA) technique, were 0.73 ng for ONA,
0.65 ng for ABO, and 0.44 ng for INCO (Table 1)[32].
The specific neurotoxin potency or biological activity
(U) per mass of neurotoxin protein was calculated based on
the overall mean concentration of BoNT/A neurotoxin,
giving INCO the highest specific biological activity (U/ng
neurotoxin) at 227 U/ng compared with 137 U/ng for ONA
and 154 U/ng for ABO [32,46]. INCO contains no other
clostridial proteins, and, therefore, the specific biologic
potency relative to the total foreign protein is 227 U/ng. As
the reported clostridial protein content per 100 U of ONA
is 5 ng and of ABO is 4.35 ng, the equivalent specific
biologic potency relative to the total foreign-protein load
for onabotulinumtoxinA is 20 U/ng and for ABO is 115 U/
ng. Thus, the foreign-protein load delivered per unit of
INCO is lower than that for both ONA and ABO.
The units of ABO are different from those of ONA and
INCO. However, comparing ONA and INCO, which have
demonstrated similar clinical activity [32], the findings
suggest that 0.44 ng of INCO has the same biological
activity as 0.73 ng of ONA. It is hypothesized that part of
the neurotoxin in ONA may be inactive or denatured due to
the specific vacuum-drying process used in the manufac-
ture of the final drug [32,49].
7 Spread and Diffusion
Discussions on neurotoxin spread and diffusion are ham-
pered by inconsistent use of terminology. Spread occurs
when the injected molecule travels from the original
injection site, for example as a result of injection tech-
nique, volume of injection, or needle size. In contrast,
diffusion indicates the passive movement of neurotoxin
toxin along a concentration gradient within the target tissue
beyond its original injection site [50].
Precise localization of neurotoxin is required to produce
the desired clinical results. Temporary disfigurement or
functional impairment can occur if the neurotoxin diffuses
into adjacent muscle. Aoki et al. [51] proposed that dif-
ferent diffusion characteristics were attributed to protein
complex size and pharmacological properties, whereby the
high-molecular-weight toxin complex of ONA would limit
tissue distribution and explain reported differences in side
effects favoring ONA over ABO [51]. However, more
recent studies, which have compared diffusion of BoNT/A
products by measuring the size of anhidrotic halos fol-
lowing injection of identical volumes into the forehead of
patients, suggest that this is not the case. A comparison of
ONA and ABO, using dose ratios of 1:2.5, 1:3, and 1:4,
showed that the area of anhidrosis was larger with ABO in
93 % of comparisons at all dose ratios and identical
injection volumes [52]. A separate study, which used a
dose ratio of 1:2.5, observed no significant differences
between the mean size of halos produced by the two pro-
ducts [53]. There were no differences in product diffusion
when the same dose was injected with the same technique.
A comparison of INCO with ONA showed no difference in
the size of the anhidrotic area produced following injection
of 5 U of INCO versus 5 U of ONA on either side of the
forehead after 6 weeks and 6 months. Importantly, the
adverse event profile in the pivotal head-to-head studies did
not show any difference between INCO and ONA [8–10].
While containment of diffusion is a desirable goal [54],
data show that the presence of complexing proteins in the
pharmaceutical preparation does not reduce migration of
the neurotoxin [55].
The underlying reason for the lack of difference in
diffusion is because the neurotoxin is already dissociated
from the complexing proteins after reconstitution of a vial
prior to injection into target tissues, and migrates alone in
the injected tissue [31].
4 J. Frevert
8 Stability
In the commercial formulations evaluated in this paper,
human serum albumin (HSA) is required to stabilize the
BoNT/A products, with ABO having the lowest content of
all (Table 1)[1–6]. The low amount of HSA in ABO could
at least partly explain why not all the neurotoxin in ABO is
bioavailable depending on the concentration of the HSA in
the injected volume [56]. According to respective product
labels, ABO has a shelf life of 2 years at 2–8 °C, ONA can
be stored for 2 or 3 years at 2–8 °C (depending on the
number of units) or in the freezer, and INCO has a shelf life
of 3 or 4 years at room temperature. After reconstitution,
ONA and INCO are stable for 24 h at 2–8 °C, and ABO is
stable for 4 h at 2–8 °C[1–6]. The prolonged shelf life and
less stringent temperature restrictions displayed by INCO
(Table 1) suggest that complexing proteins are not required
for BoNT/A stability [57]. Among the three leading
available BoNT/A products, INCO is the only botulinum
product that is stable in lyophilized form for up to 4 years
at room temperature, whereas ONA and ABO products
must be stored refrigerated [58]. In a stress stability study,
INCO survived storage at temperatures as high as 60 °C for
1 month without loss of potency [57].
9 Immunogenicity
Immunogenicity refers to the ability of a protein product to
elicit antibody formation. As with any therapeutic protein,
botulinum toxin is regarded as foreign by the host and
therefore has the potential to induce an immune response,
particularly with repeated administration. This can lead to
the development of neutralizing antibodies that may or may
not result in secondary treatment failure. Overall, BoNT/A
products exhibit lower clinically detectable levels of anti-
bodies than do other approved biologic products [59]. The
development of neutralizing antibodies is more common in
therapeutic indications, where doses tend to be much lar-
ger, but they are increasingly been reported in patients
receiving botulinum toxin for aesthetic treatment along
with cases of secondary non-responsiveness [60–64]. A
number of factors can impact the immunogenicity of bot-
ulinum neurotoxins, including product-related factors such
as the manufacturing process, the antigenic protein load,
and the presence of complexing proteins, as well as treat-
ment-related factors such as the overall toxin dose, booster
injections, and prior exposure.
A distinguishing feature among the commercially
available neurotoxins is the presence or absence of com-
plexing proteins. NAPs do not play a role in toxin-induced
blockade of cholinergic transmission and, until recently,
were thought to be just a group of passive bystanders when
injected for therapeutic and aesthetic uses. However, sev-
eral lines of evidence that have examined the fate and
possible interactions of NAPs with patient tissues after
intramuscular injection suggest this may not be the case
and that the presence of complexing proteins might be
clinically relevant [65–67].
Preclinical data have shown that, in contrast to ONA and
ABO, INCO does not lead to the production of neutralizing
antibodies following repeated injections into New Zealand
white rabbits [65]. Kukreja et al. [66] measured the
immunological reactivity of BoNT/A in its purified and
complex forms and demonstrated that BoNT/A with com-
plexing proteins (including HA-33) triggered a stronger
immune response than the purified 150 kDa neurotoxoid
alone. HAs are known to act as adjuvants [68,69] and can
bind and activate dendritic cells, which play a key role in
early phases of the immune response. In particular, HA-33
is the largest component of the complexing proteins and a
major immunoreactive protein in the BoNT/A complex
[70,71].
That complexing proteins can induce an inflammatory
response has recently been demonstrated in a human neu-
roblastoma cell line (SH-SY5Y) [67]. While pure BoNT/A,
BoNT/A complex, and NAPs all bound to the SH-SY5Y
neuronal cells, the BoNTA complex and NAPs additionally
bound to lymphoblasts and fibroblasts. Furthermore, pure
BoNT/A did not affect inflammatory cytokine release,
whereas the BoNT/A complex and NAPs increased the
release of multiple inflammatory cytokines. Moreover, the
cytokines induced by the BoNTA complex and by NAPs
alone varied, suggesting that the different structure of
BoNT/A complex induces significantly differential host
response in human neuronal cells.
The clinical implication is that complexing proteins are
immunogenic and can elicit an immune reaction against
BoNT/A [67]. However, antibody titers required to cause
resistance to botulinum toxin have not been defined and
immune responses can differ between patients. Further-
more, variability in the reported prevalence of neutralizing
antibodies and treatment failure can be attributed to study
design, administered doses, indication, assay methodology,
timing of serum sample testing, and treatment history [72,
73]. Not all immune responses preclude the biological
therapy from being clinically effective. Only antibodies
that bind botulinum toxin in a manner that neutralizes its
biological activity sufficiently will attenuate its effect on
the neuromuscular junction. Thus, the formation of anti-
bodies may have no effect on treatment or may result in
partial or complete clinical unresponsiveness to botulinum
toxin type A [74,75]. However, there is a risk that antibody
titers will increase with further injections, which might
have a booster effect. Today’s cosmetic patients start their
aesthetic treatments at increasingly younger ages and not
Pharmaceutical, Biological, and Clinical Properties of BoNT/A Products 5
only for a single indication, resulting in an increased fre-
quency of neurotoxin use, as well as a larger total amount
of neurotoxin use over a lifetime.
The prevalence of patients developing neutralizing
antibodies after long-term treatment with ONA or ABO is
dependent on the condition being treated and thus treat-
ment dose, with incidence rates ranging from 0.3 to 6 %
[72,76–82]. To date, there has been only one case of
antibody-induced therapy failure with INCO. This occurred
in a patient with progressive hereditary juvenile-onset
generalized dystonia whose immune system had already
been sensitized by pretreatment with ABO for 15 years
[83], supporting the hypothesis of reduced immunogenicity
with INCO [84]. Furthermore, a prospective blinded study
in 37 cervical dystonia patients previously treated with
ONA or ABO who developed neutralizing antibodies and
partial secondary non-responsiveness, reported that con-
tinuous treatment with INCO every 3 months for
48 months did not result in an increase in neutralizing
antibody titer [85]. Despite a transient increase in ten
patients in the first 24 months, neutralizing antibodies in
fact declined significantly below the initial titer in 84 % of
patients (P\0.001), and 62 % of patients became
seronegative.
In addition to selecting a product with a low risk of
antigenicity, it is important to establish good practice to
minimize the risk of neutralizing antibodies. Studies of
BoNT/A formulations containing complexing proteins
suggest that higher dosing frequency, short treatment
intervals, and greater number of injections may increase
the likelihood of their development [75,86–88]. Most
experts currently recommend using the smallest dose that
achieves the desired clinical effect, avoiding booster
injections, and waiting at least 3 months between
treatments.
10 Conclusions
The repetitive contraction and activity of the muscles
involved in facial expression is a major factor in the for-
mation of lines and wrinkles, especially in the forehead and
around the eyes. Botulinum toxin blocks presynaptic ace-
tylcholine release, thus preventing the nerve impulses
responsible for muscle contraction, and can be used to treat
all wrinkles that are the result of normal facial movement.
Practitioners currently have a choice of three BoNT/A
products for the treatment of facial lines. As of 2014, ONA
and INCO share the same aesthetic indications: the tem-
porary improvement in the appearance of moderate to
severe glabellar lines and crow’s feet lines (the latter
indication is approved in Europe, but not yet in the USA) in
adults younger than 65 years of age [2,3]. ABO currently
only has aesthetic approval for the treatment of moderate to
severe glabellar lines [4]. However, all three products are
effectively used off-label for a number of other aesthetic
indications.
ONA and INCO have comparable efficacy, with a 1:1
conversion ratio, and have demonstrated therapeutic
equivalence in different indications, including cervical
dystonia, blepharospasm, glabellar lines, and crow’s feet.
The ONA to ABO conversion ratio is approximately 1:2.5.
All three preparations have similar mechanisms of
action. For storage stability and convenience of handling,
BoNT/A products are formulated as either lyophilized
(ABO and INCO) or vacuum-dried powders (ONA). Any
one of the pH, temperature, formulation, and concentration
range conditions required to lyophilize or vacuum dry a
botulinum toxin into a format ready for reconstitution by a
physician can increase the likelihood of inactivated toxoid
proteins that may be immunogenic. Of the three products,
ONA is the only one dissolved in a solution containing
NaCl prior to drying, which has been proposed as a
potential explanation for its toxoid content [32,41]. The
presence of inactive botulinum toxin molecules in a clinical
preparation will contribute to the overall protein load of the
preparation without contributing to its clinical efficacy.
The major difference between the three products relates
to the presence or absence of complexing proteins. INCO
consists of only the pure neurotoxin and contains no other
clostridial proteins. Its foreign-protein load delivered per
unit of toxin is lower than that for both ONA and ABO.
Complexing proteins are not required for the effectiveness
of BoNT/A preparations for injection. They are not
required for the stability of BoNT/A preparations, nor do
they limit their diffusion, and a definitive need for the
presence of NAPs in therapeutic and aesthetic indications
has not been established. Until recently, much of the
information surrounding NAPs was speculative, but data
are beginning to emerge that show that complexing pro-
teins, and in particular HAs, can trigger an immune
response.
The therapeutic benefits of BoNT/A are not permanent,
and periodic injections are necessary. While immunoge-
nicity may not yet be a major issue in aesthetic indications
because of the low doses used, the concern is that it may
become one in subjects receiving frequent dosing over a
prolonged period; for example, an individual who begins
treatment for glabellar lines and crow’s feet at 30 years of
age and who receives repeat injections several times a year
over the next 35 years. Given the lack of therapeutic effect
of NAPs for therapeutic and aesthetic indications, clinical
strategies to reduce or eliminate neutralizing antibody
development and secondary treatment failure are warranted
and include using the lowest effective dose, with the lon-
gest acceptable interval between injections.
6 J. Frevert
Acknowledgments Dr. Vanessa Gray-Schopfer, OmniScience SA,
provided medical writing services funded by Merz Pharmaceuticals,
GmbH, Germany. The author was fully responsible for the content
and editorial decisions of this manuscript. Dr. Ju
¨rgen Frevert is an
employee of Merz Pharmaceuticals, GmbH, Germany. The author
wishes to acknowledge the contribution of Jenny Grice for helping to
finalize this manuscript. This activity was supported by an unre-
stricted educational Grant provided by Merz Pharmaceuticals GmbH.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and the source are credited.
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