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Citation: Rasetti-Escargueil, C.; Palea,
S. Embracing the Versatility of
Botulinum Neurotoxins in
Conventional and New Therapeutic
Applications. Toxins 2024,16, 261.
https://doi.org/10.3390/
toxins16060261
Received: 15 April 2024
Revised: 26 May 2024
Accepted: 27 May 2024
Published: 4 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
toxins
Review
Embracing the Versatility of Botulinum Neurotoxins in
Conventional and New Therapeutic Applications
Christine Rasetti-Escargueil 1, * and Stefano Palea 2
1The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
2Humana Biosciences-Prologue Biotech, 516 Rue Pierre et Marie Curie, 31670 Labège, France;
stefano.palea@humana-biosciences.com
*Correspondence: christine.rasetti-escargueil@icr.ac.uk
Abstract: Botulinum neurotoxins (BoNTs) have been used for almost half a century in the treatment
of excessive muscle contractility. BoNTs are routinely used to treat movement disorders such as
cervical dystonia, spastic conditions, blepharospasm, and hyperhidrosis, as well as for cosmetic
purposes. In addition to the conventional indications, the use of BoNTs to reduce pain has gained
increased recognition, giving rise to an increasing number of indications in disorders associated with
chronic pain. Furthermore, BoNT-derived formulations are benefiting a much wider range of patients
suffering from overactive bladder, erectile dysfunction, arthropathy, neuropathic pain, and cancer.
BoNTs are categorised into seven toxinotypes, two of which are in clinical use, and each toxinotype
is divided into multiple subtypes. With the development of bioinformatic tools, new BoNT-like
toxins have been identified in non-Clostridial organisms. In addition to the expanding indications of
existing formulations, the rich variety of toxinotypes or subtypes in the wild-type BoNTs associated
with new BoNT-like toxins expand the BoNT superfamily, forming the basis on which to develop
new BoNT-based therapeutics as well as research tools. An overview of the diversity of the BoNT
family along with their conventional therapeutic uses is presented in this review followed by the
engineering and formulation opportunities opening avenues in therapy.
Keywords: botulinum neurotoxins; toxinotypes; subtypes; engineering; therapy
Key Contribution: An overview of the wide diversity of the BoNT family along with their conven-
tional therapeutic uses is presented in this review followed by the engineering techniques and new
formulation opportunities opening new avenues in therapy.
1. Introduction
The first clinical applications of BoNTs were envisioned by Dr Justinus Kerner in 1815–
1817 when he proposed the use of this “sausage toxin” to treat hypersecretion disorders.
Dr Justinus Kerner was a German physician (as well as Romantic poet and polymath) who
established the link between flaccid paralysis and the consumption of spoiled sausages. Dr
Kerner had investigated numerous “sausage poisoning” cases following blood sausages
consumption in the southwest region of Germany. He then established that an unknown
toxin, able to develop under anaerobic conditions in the blood sausages was lethal in minute
doses by causing descending flaccid paralysis. Dr Kerner formulated for the first time the
therapeutic potential of this sausage poison thanks to its ability to inhibit muscle tonicity
and alleviate hypersecretion or neurological disorders resulting from muscle overactivity.
In 1895, Van Ermengen identified the causative agent, Bacillus botulinum, at this stage
renamed the “Clostridium botulinum”, isolated during a severe outbreak that killed three
musicians in a Fanfare in Belgium, while many others became seriously ill. To complete his
findings, Van Ermengen found that the culture filtrates administered to animals induced
botulism and led to death [
1
–
3
]. Nevertheless, it took almost a century before Dr Alan Scott
Toxins 2024,16, 261. https://doi.org/10.3390/toxins16060261 https://www.mdpi.com/journal/toxins
Toxins 2024,16, 261 2 of 29
discovered the benefit of botulinum toxin (Oculinum©) to treat strabismus [
4
]. Nowadays,
a wide range of potential therapies remain unexploited in complement to the present
formulations exclusively based on the first two identified toxinotypes, namely BoNT/A
and BoNT/B. Furthermore, genetically engineered botulinum toxin products are now in
development to further expand therapeutic uses [5].
BoNTs form a family of neurotoxins produced by spore-forming Gram-positive anaero-
bic bacteria named Clostridium botulinum and Clostridium spp., such as Clostridium butyricum,
Clostridium argentinensis, and Clostridium baratii [
6
,
7
]. Botulinum neurotoxins are part of the
“dirty dozen” agents listed as bioweapons by the CDC [
8
]. All Clostridium botulinum strains
were historically classified into four distinct metabolic groups (I–IV) according to their
biochemical properties and their physiology: saccharolytic and proteolytic abilities, alcohol
fermentation products, heat resistance of their spores, and ability to grow in acids, salts,
and alcohol at different temperatures [
3
,
9
]. Rare but often severe intoxication by BoNTs, or
toxi-infection by these bacteria, results in a clinical condition called botulism, or intestinal
botulism, due to intestinal colonisation by Clostridium spores, respectively. Flaccid paralysis
may be fatal if the patient has no access to intensive care. Unfortunately, the treatment of
botulism nowadays remains mainly symptomatic, including assisted ventilation within the
intensive care unit for severe cases.
BoNTs are 150 kDa modular proteins consisting of two peptide chains connected by
a disulfide bond: a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC) [
10
]. The
translocation domain consists of a 50 kDa chain at the N-terminal domain (HN). This
domain is involved in the translocation of the LC, containing a metalloprotease, into the
intracellular compartment, after the receptor binding domain of the BoNT has bound
to an outer membrane receptor of the target neuronal cell [
11
,
12
]. The peptidic chains
of BoNTs present significant differences along their amino acid sequences among each
subtypes [
13
]. The C-terminal domain (HC), is composed of two sub-domains (HCn and
HCc). This HC domain is involved in the binding of the toxin to neuronal membranes
at specific presynaptic receptor sites. The delivery of the LC into neuronal cells through
the vesicle membranes is subsequently mediated by the translocation domain situated on
the N-terminal domain of the heavy chain (HN) to facilitate its entry into the neuronal
cytosol. The HN is a helical bundle that protects and escorts the LC across endosomal
membranes. This protein-conducting channel is actioned by the transmembrane proton
gradient that supports LC cargo unfolding during translocation and finally triggers the
release and refolding of the LC in the cytosol [
14
,
15
]. The modular architecture of BoNT
is such that each BoNT module functions individually while each domain serves as a
chaperone for the others. The receptor binding domain facilitates the unfolding of the
LC to reach a translocation permissive conformation in synchrony with the formation of
the translocation channel formation. The modular function of the translocation domain
makes BoNT an attractive tool for molecules delivery in target tissues. By replacing the
neuronal targeting binding domain with one domain recognising a specific surface protein
could transform the BoNTs into delivery system targeting specific tissues [
16
]. The HCn
subdomain is involved in toxin binding by interacting with lipid microdomains [17–19].
The BoNT holotoxins are produced as non-covalently bound complexes of different
protein components forming the progenitor toxin complexes (PTCs) [
20
]. The progenitor
toxin complex is composed of non-toxic neurotoxin-associated proteins (NAPs) such as
hemagglutinins (HA-17, HA-33, and HA-70) and a non-toxic non-hemagglutinin (NTNHA)
protein. These auxiliary proteins (HAs and NTNHAs) ensure appropriate stabilisation,
preservation, and absorption of the BoNTs during the intoxication process [21–23].
BoNTs are initially produced as single chain proteins undergoing post-translational
proteolytic activation via environmental or host proteases into a disulfide-linked ~50 kDa
light chain (LC) and ~100 kDa heavy chain (HC) di-chain protein before reaching the
host [
24
,
25
]. BoNT/E is produced as a single chain toxin and later cleaved by the host
proteases into the di-chain form which is about 100-fold more toxic than the single chain
Toxins 2024,16, 261 3 of 29
form. This cleavage step shows that di-chain formation is an essential step of the BoNT
activation process [26].
The BoNTs enter synaptic terminals and cleave one of the three soluble N-ethylmaleimide-
sensitive factor attachment protein receptor (SNARE) proteins, namely vesicle-associated
membrane protein (VAMP), synaptosomal-associated protein 25 (SNAP25), and syntaxin,
leading to the inhibition of neurotransmitter release by synaptic vesicles [
14
]. Botulism
can last for months and require extensive medical treatment. The only available therapy
is antitoxin administration, only effective if administered early before toxin entry into the
neuronal cells [
27
]. Besides this shared mechanism of neurotransmitter release disruption,
the intracellular targets and receptors, the pharmacodynamic/kinetic properties greatly
vary between the various BoNT toxinotypes.
BoNTs are categorised into at least seven toxinotypes, termed A–G, two of which are
in clinical use, types A and B. Each toxinotype is divided into multiple subtypes using
a number defined by amino acid sequence differences >2.6% [
28
]. In 2014, an eighth
novel BoNT, called BoNT/H, was identified by Arnon and colleagues in clinical isolates,
on the basis of sequence analysis and its lack of neutralisation by sera against known
toxinotypes [
29
]. Nevertheless, this toxinotype was later identified as BoNT/FA or HA
mosaic toxin, named BoNT/H or BoNT/FA, since its mosaic structure comprised regions
of similarity with BoNT/A and F and BoNT/A antitoxin was able to neutralise it. Its
LC showed similarities to BoNT/F5 and the HC presented similarities with BoNT/A1-
HC [30,31].
More recently, with the development of bioinformatic tools, new BoNT-like toxins have
been reported and characterised, including BoNT/X in a Clostridium botulinum strain [
32
,
33
].
New BoNT/like sequences were identified in non-clostridial species such as Weissella
oryzae and Chryseobacterium piperi, BoNT/En protein was found in an Enterococcus faecium
strain [
33
], Paraclostridial Mosquitocidal Protein 1 (PMP1) was found in the Paraclostridium
bifermentans strains [
34
], and PGT1/2 protein was found in Paeniclostridium ghonii [
35
].
Among those BoNT-like proteins, BoNT/X cleaves VAMP1-3 at a unique site, and it cleaves
VAMP 4 and 5, as well as Ykt6 (a VAMP family protein). LC/X is also ~10-fold more
efficient at the cleavage of VAMP1 [
26
,
36
,
37
]. However, the native BoNT/X produced by C.
botulinum Strain 111 and the recombinant form of BoNT/X exhibit very weak potency in
human-induced pluripotent stem cell-derived neuronal cells and in the mouse model [
38
].
The other non-clostridial LC/En cleaved VAMP 1-3, Syntaxin (Syx 1B and Syx 4), as well
as SNAP-23 and SNAP-25 but at lower efficiency for SNAP-25, Syx 1B, and Syx 4 [
26
].
The LC/Wo cleaves VAMP-2 [
39
]. Understanding the complexity of BoNTs and BoNT-
producing clostridia nomenclature represents one crucial challenge to ensure accurate
reporting in diagnostic settings, in food safety testing and evaluation of therapeutic BoNTs.
For example, it is paramount to distinguish between Botulinum strains associated with
a human botulism case, an animal outbreak, a food safety risk, or BoNT therapeutic
formulation analysis [40].
Because of the known variability of BoNT-derived products, a summary of the ther-
apeutic profiles of the conventional BoNT formulations will be presented in this review,
in parallel to more recent formulations involving engineered BoNTs and exploiting the
versatility of the BoNTs to develop new injection-free or local formulations.
2. Overview of Conventional Therapeutic Applications and Formulations
A few years after Dr Alan Scott’s discovery showing that BoNT/A could relax eye
muscles and treat strabismus, the first approved BoNT/A formulation was licensed in 1984
for human therapeutic use.
In the 1970s, corrective surgery to treat strabismus required improvements since most
patients needed reoperation. Dr Scott, an ophthalmologist, was testing substances able to
weaken eye muscles and realign them. To this aim, he experimentally injected Botulinum
toxin A into eye muscle on monkeys. He found that muscle weakness induced by BoNT/A
was specific and prolonged, causing no local side effects nor systemic toxicity [
41
]. He
Toxins 2024,16, 261 4 of 29
then formulated the Botulinum toxin A produced by Dr. Edward Schantz, who was a
microbiologist in the Johnson laboratory at the University of Wisconsin, to become a safe
formulation for injection into human patients. The toxin stabilising agent was changed
from the gelatin to human serum albumin since it was approved for human use. Dr Scott’s
team soon established that the toxin amounts were to be expressed based on the mouse
LD50 test to assess its actual biological activity rather than measure a mass of protein.
After successfully treating strabismus in humans, Dr Scott and his collaborators used
BoNT/A to treat blepharospasm and torticollis patients. Subsequently, Dr Joseph Tsui, a
neurologist, injected the toxin to treat multiple sclerosis patients presenting with spasticity.
The product from the Schantz and Johnson laboratory was registered and manufactured
until the late 1970s through the Oculinum company under the name Oculinum
®
[
42
].
Oculinum
®
was subsequently distributed by Allergan, Inc. to meet the growing clinical
demand [
43
]. The beneficial effects of BoNT/A on facial glabellar lines were observed by
chance when ophthalmologists treated patients suffering from involuntary blinking. The
Allergan company modified the brand name of Oculinum
®
to Botox
®
after the finalisation
of the acquisition of Oculinum, Inc. company in 1991.
Meanwhile, the Public Health Laboratory Service in the United Kingdom developed a
different BoNT/A product. In 1992, this BoNT/A product was licensed for Europe with
the brand name Dysport
®
and non-proprietary name “abobotulinumtoxinA”. Then, Ipsen
France acquired the company and subsequently sold the cosmetic operations to Galderma
(Lausanne, Switzerland) who renamed the Dysport
®
product as Azzalure
®
[
44
,
45
]. Given
that only minute doses of BoNTs are sufficient to silence neuromuscular junctions as
well as modulate sensory fibres, natural BoNTs represent an ideal therapeutic repertoire.
Moreover, only a small proportion of patients may develop resistance due to the production
of antibodies following repeated injections of BoNT. In addition to its extreme potency, the
effects of BoNT/A can last for more than 3–6 months in humans.
2.1. A Wide Range of Conventional Therapeutic Applications
Dr. Jankovic, who was also a pioneer in using BoNT injections into patients to treat
blepharospasm, led and published the first double-blind, placebo-controlled trial focused
on the treatment of cranial–cervical dystonia by BoNT [
46
]. Those trials were instrumen-
tal to obtain approval by the Food and Drug Administration (FDA) in 1989 for BoNT
formulations to treat blepharospasm, hemifacial spasm (HFS), and other types of facial
nerve disorders [
47
]. BoNTs have been given regulatory approval for the treatment of
many disorders caused by muscle over-contractility. Therefore, BoNTs are routinely used
to treat movement disorders like dystonia or spasticity. Regarding movement disorders
originating from neurologic diseases, BoNT was proven to be effective in patients with
bruxism, tremors, tics, myoclonus, restless legs syndrome, and tardive dyskinesia. BoNT
has been shown to be effective against common disorders observed in Parkinson’s disease
such as foot dystonia, freezing of gait, rigidity, and tremor [45].
Recent studies have expanded the use of BoNT from a powerful muscle relaxant in the
periphery to the relief of neurodegenerative disease originating from the central nervous
system. In addition to muscle hyperactivity, BoNTs are also efficient to treat hypersecretion
disorders like hyperhidrosis and sialorrhea [48,49].
Furthermore, BoNTs represent a unique therapeutic opportunity to modulate neuronal
function since they are reported to affect inhibitory, excitatory, and sensory neurons [
50
].
Experimental observations show that BoNT is involved in the modulation of sensory feed-
back loop going to the central nervous system and resulting in analgesia. The therapeutic
benefit of BoNT has been shown in chronic migraine [51]. As a result, the use of BoNTs to
reduce pain has increased significantly, suggesting an expanding range of applications in
chronic pain. BoNTs have been shown to inhibit the release of glutamate, substance P (SP),
and calcitonin gene-related peptide (CGRP) acting as pain neurotransmitters by altering
synaptic vesicle fusion and transient receptor potential (TRP) channels at the neuronal
membrane [
52
]. Dr Aoki and coworkers established that nociception inhibition by BoNT/A
Toxins 2024,16, 261 5 of 29
was due to the alteration of the mechanosensitive ion channels fusion into the nerve termi-
nals of peripheral trigemino-vascular neurons. In the treatment of migraine headache, the
action of BoNT/A is due to the blockade of neurotransmitters release as well as inhibition
of inflammatory peptides release and relevant cell surface ion channels expression [53].
Besides their targeting of neurons, the effects of BoNTs were evidenced in human
skin restoration and other tissues, which will expand the therapeutic uses of BoNTs [
54
].
Studies showing that BoNT/A can induce skin cell restoration and improve global skin
condition augur a wider impact of BoNTs than exclusive SNARE cleavage at neuronal
vesicles. A range of experimental studies show the protective effects of BoNTs on skin flaps,
wound healing, hypertrophic scars, and psoriasiform dermatitis, and wider effects like anti-
inflammatory and anti-cancer effects [
54
]. Injections of BoNT-A have been shown to reduce
signs and symptoms of acne, rosacea, and psoriasis and to bring about significant improve-
ments in several rare diseases that are caused or exacerbated by hyperhidrosis [
55
]. More
recently, experimental studies have evidenced the beneficial effects of BoNT/A in alopecia
after artificially inducing hair loss in the mouse under continuous stress conditions [56].
2.2. Variability in the BoNT Formulations
The available therapeutic formulations of BoNT currently approved for use in hu-
man in the USA and in Europe contain only the two main toxinotypes of BoNT, namely
botulinum toxin type A (BoNT/A) or botulinum toxin type B (BoNT/B): abobotulinumtox-
inA (ABO, Dysport
®
product from Ipsen, Les Ulis, France), incobotulinumtoxinA (INCO,
Xeomin
®
or Bocouture
®
products from Merz Pharmaceuticals GmbH, Frankfurt, Germany),
onabotulinumtoxinA (ONA, Botox
®
product from Abvie/Allergan, Irvine, CA, USA), and
rimabotulinumtoxinB (Myobloc
®
from Solstice Neurosciences now Supernus Pharmaceu-
ticals, Rockville, MD, USA) (see Table 1) [
57
]. Currently, BoNTs are employed for an
expanding number of indications associated with muscle overactivity and for cosmetic
purposes, in more than 20 therapeutic formulations for almost 20 therapeutic indications
(see Table 1for timelines of approval of each formulation). The following ratios were
calculated, based on initial studies, in order to achieve a similar efficacy in clinical practice:
onabotulinumtoxinA versus incobotulinumtoxinA = 1; onabotulinumtoxinA versus abobo-
tulinumtoxinA = 1:2.5; onabotulinumtoxinA versus rimabotulinumtoxinB = 1:50; these
ratios show that the different BoNT formulations are not interchangeable [
58
]. A significant
number of clinical studies have confirmed that ONA and INCO show therapeutic equiva-
lence in a wide range of indications notably cervical dystonia and blepharospasm, whereas
between ONA and ABO formulations, the application of a predetermined conversion ratio
(close to 1:25) was recognised as being the most appropriate [59].
New BoNT formulations are under development, but with only a few being FDA-
approved thus far. DaxibotulinumtoxinA (Daxxify
®
) is a new formulation of BoNT/A for
cervical dystonia which showed a median effect duration of 24 weeks at doses of 125 U.
The DaxiBoNT-A formulation contains a stabilising peptide that ensures longer-lasting
effects and a shelf-life of two years. The DaxiBoNT-A formulation does not contain animal-
derived components nor human albumin and does not require refrigeration. The evaluation
of the safety, duration of response, and efficacy of two doses of DaxibotulinumtoxinA
for the treatment of cervical dystonia (CD) served as the foundation for the Food and
Drug Administration (FDA) approval of Daxxify
®
in August 2023 [
60
,
61
]. This prolonged
duration of action represents a significant step forward from previous formulations since
this will allow a longer inter-visit interval than the usual interval of about 3 to 4 months.
However, it should be mentioned that the prolonged effect may also be due to the higher
doses of Daxxify
®
injected. The LanbotulinumtoxinA (Lantox
®
product, Lanzhou, China)
is a more classical formulation of BoNTA manufactured and registered in China since 1994.
Despite its widespread use in China and other Asian countries and in South America, it is
not yet approved elsewhere [62].
Toxins 2024,16, 261 6 of 29
Table 1. History of botulinum neurotoxin (BoNT) research and clinical development.
1822 Justinus Kerner Sausage poison (envisioned therapeutic potential)
1870 Müller Disease called “botulism” (Latin: botulus meaning sausage)
1895 Van Ermengem Clostridium botulinum (microorganism causing botulism)
1919 G.S. Burke Minimum lethal dose established in guinea pigs
1928 Herman Sommer Isolation and purification of BoNT
1946 Carl Lamanna
Edward Schantz
Neurotoxin activity determined using the LD50 test
BTA produced in the crystalline form
1949 Arnold Burgen BoNT induces the blockade of the
neuromuscular transmission
1950 Vernon Brooks BoNT/A: -blockade of acetylcholine from motor
nerve endings
1960s Schantz/Scott BoNT/A to treat strabismus in monkeys
1980 Scott BoNT/A to treat strabismus in humans
1987 Drs. Jean and
Alastair Carruthers
Ophthalmologists treating patients for involuntary blinking
discover the cosmetic benefits of BoNT/A
1988 Allergan First clinical trial on Oculinum (BoNT/A)
1989 FDA Allergan
First official indications: strabismus, blepharospasm,
hemifacial spasm, and dystonia
Allergan buys and renames Oculinum as Botox®
1990 MHRA Dysport®(abobotulinumtoxinA, Ipsen) (~) approval
for dystonia
1993 Montecucco and
Schiavo SNAP-25 is the molecular target of BoNT/A
1995 MHRA Dysport®(abobotulinumtoxinA, Ipsen, Wrexham, UK)
approval for strabismus in the UK [63]
1997 China
Approval of Lantox®(lanbotulinum toxin A, Lanzhou
Institute of Biological Products, Lanzhou, China) for
strabismus, blepharospasm, and hemifacial spasm.
1999 FDA (New) Botox®(onabotulinumtoxinA, Allergan, Irvine, CA,
USA) approval for cervical dystonia
2000 FDA First BoNT/B: NeuroBloc®approved for cervical dystonia
2002 FDA
Botox®approved for cosmetic uses as Botox Cosmetics®
(Australia, Switzerland, Taiwan, and Singapore)
Lantox®approval in Republic of Korea
2003 AFSSAPS Botox product approved under Vistabel®name (France)
2004 FDA Botox®approved for primary axillary hyperhidrosis
2005 EMA
Xeomin
®
(incobotulinumtoxinA, Merz, Frankfurt, Germany)
approved for blepharospasm and cervical dystonia in adults
(Germany).
2006 MHRA Botox®approved under the name Vistabel®for
glabellar lines
2006 Korean FDA
Neuronox®(MedyTox, Seoul, Republic of Korea) approved
for blepharospasm
(Meditoxin®, Botulift®, Cunox®)
2007 India Approves Boto genie®(Bio-med Ltd., Ghaziabad, India)
Toxins 2024,16, 261 7 of 29
Table 1. Cont.
2009 MHRA
FDA
Azzalure®approval for treatment of glabellar lines
Dysport®(Abobotulinum) approved for glabellar lines and
cervical dystonia
2010 FDA
Botox®approval for adult upper limb spasticity
Xeomin
®
(Incobotulinum) approval for cervical dystonia and
blepharospasm
2011 FDA
Botox®approval for chronic migraine and
urinary incontinence
Xeomin®(incobotulinumtoxinA) approval as Bocouture®
for glabellar lines in adult patients
2012 NHS UK
China
Botox®approval for chronic migraine
Lantox®approval for glabellar frown lines
BoNT/A-specific cell-based potency assay to replace the
mouse bioassay [64]
2013
Korean FDA
FDA
Japan
Nabota
®
(Daewoongs Pharmaceuticals, Hwaseong, Republic
of Korea) approval
Evosyal®, Daewong/Evolus-Alphaeon R. Korea/USA
Botox®approval for overactive bladder
Nerbloc®(Eisai, Tokyo, Japan) approval
2014 China
Korea
CBTX-A approved as Lantox®and Prosigne®(Lanzhou
Institute of Biological Products, Lanzhou, China) (Hengli®,
Lanzox®, CBTX-A®, Redux®, Liftox®, Dituroxal®)
Botulax
®
, Zentox
®
, Regenox
®
(Hugel Pharma, Gangwon-do,
Republic of Korea)
2015 FDA
EMA
Xeomin®(incobotulinumtoxinA) and Dysport®
(AbobotulinumtoxinA) approval for adult upper
limb spasticity
Approves Bocouture®for combined upper facial lines
2016 Korean FDA
FDA
Approval of Coretox
®
(Medytox, no complexing proteins, no
biological excipients)
Approval of Dysport®for children lower limb spasticity
2017 FDA and EMA,
Russia
Botox®and Dysport®approval for adult lower limb
spasticity Relatox®approval (Microgen, Moscow, Russia)
2018 FDA Approval of Botox®for forehead wrinkles and Xeomin®
for sialorrhea
2019 FDA
Approval of Botox®for paediatric upper limb spasticity
Approval of Jeuveau®(Nabota) for glabellar lines
Approval of Neuronox ®for glabellar lines
Approval of Botulax®by PDUFA (Hugel R., Gangwon-d,
Korea BT-A, Botox®analogue)
2022 FDA
Approval of RT002 (DaxibotulinumtoxinA) for glabellar lines
Revance Therapeutics (Nashville, TN, USA)
2022 Korean FDA
Masport®Masoundarou I.R. Iran BT-A, Dysport®analogue
CosmeTox®Transdermal USA BT-A, creme
Lantox®registered in Republic of Korea.
EB-001 BONTi/Allergan (Irvine, CA, USA) BT-E
2023 FDA
MCL005 Malvern Cosmeceuticals UK BT-A, topic gel
ANT-1207 Anterios/Allergan USA BT-A, lotion
DaxibotulinumtoxinA (Daxxify®, Revance, Nashville, TN,
USA) is a novel BoNTA preparation for cervical dystonia
2024 FDA
Approves Letybo
®
(Hugel, Gangwon-do, Republic of Korea)
FDA: Food and Drug administration; MHRA: Medicines and Healthcare products Regulatory Agency; AFSSAPS:
French Agency for the Safety of Health products. Modified from [62–67].
Toxins 2024,16, 261 8 of 29
All BoNT-A formulations contain the 150 kD neurotoxin with neurotoxin-associated
proteins (NAPs) except for INCO, which contains exclusively pure 150 kD neurotoxin.
Nevertheless, the production process itself may impact the active toxin quantities, such as
the addition of enzymes to increase the proportion of cleaved active toxin, which may cause
the denaturation of the neurotoxin [
68
,
69
]. As a result, the injection units are variable among
different formulations. Currently, ONA (Botox
®
) is available in 50/100/200 U vials, and
ABO (Dysport
®
) is available in 300/500 U vials. Historically, it was estimated that the initial
formulation of Botox
®
contained a dose equivalent to 25–40 ng of BNT/A for 100 mouse
LD50, while the formulation of Dysport®contained 5 ng of BoNT/A for 500 mouse LD50.
Regarding BoNT potency estimates, the variations in local BoNTs production processes as
well as variability between in-house mouse bioassays needs to be considered. Therefore,
there are differences in the pharmacodynamic and pharmacokinetic properties of the
different formulations, which cannot be used equally [70] (see Table 2).
Each BoNT/A batch produced is specific to each manufacturer, and the final formu-
lation varies between each product. While the mechanism of action of these products is
the same, there are significant formulation disparities between them. The toxin potency in
units is specific to each product, and not exactly equivalent since each company assesses
their toxin batches using its own proprietary mouse LD50 assay (or cell-based assay). As
a consequence, the “Dysport
®
unit” is not exactly equivalent to the “Botox
®
unit”, or a
“Xeomin
®
unit”. Moreover, the amount of human serum albumin also differs between the
three formulations (see Table 2), which can influence the efficacy of each formulation in
humans [
71
,
72
]. Several characteristics must be considered when administering botulinum
neurotoxins since differences occur between formulations due to diverse manufacturing
processes, bacterial strains used in fermentation, purification methods, and inactive ingre-
dients in the formulation which influence the potency and antigenicity of the products. A
range of clinical studies showed that the therapeutic and safety margins for Botox
®
were
larger than for Dysport
®
, due to a reduced tendency for Botox
®
to cause undesired distant
effects through diffusion related to its lower dosage. The adjustment in injection volumes,
techniques, and patterns are required to achieve similar clinical results [
72
]. In addition to
the variability between dose units, new formulations such as Prabotulinumtoxina A-xvfs,
Neu-botulinumtoxinA (NeuBoNT-A), Letibotulinumtoxin A, botulinum toxin E (rBoNT-E),
Innotox, and QM-1114 (Galderma) are being studied in clinical trials for different conditions.
DaxiBoNT-A, NeuBoNT-A, and rBoNT-E are in clinical trials for potential indication in
patients presenting with dystonia [
67
,
73
] (see Table 3). In practice, while being considered
as therapies for disorders related to dystonia, there is critical lack of data regarding off-label
uses. Additional clinical trials are required to expand the therapeutic applications of BoNT
toward less conventional clinical applications. Considering the diversification of the new
BoNT formulations currently in development, the therapeutic applications will certainly
grow significantly in the near future [74].
For those reasons, practice guidelines were published by the American Academy of
Neurology (AAN) in 2016 regarding the injection of botulinum toxin (BoNT) in the treat-
ment of blepharospasm, cervical dystonia, adult spasticity, and headache, and were applica-
ble to the four commercially available botulinum toxin formulations OnabotulinumtoxinA
(ONA), AbobotulinumtoxinA (ABO), IncobotulinumtoxinA (INCO), and Rimabotulinum-
toxinB (RIMA). Guidance related to BoNT treatment was also included in the guidelines
issued by the European Federation of the Neurological Societies (EFNS) focused on the
treatment of primary dystonia [75].
While BoNT uses in blepharospasm and cervical dystonia are FDA-approved, BoNT
is administered routinely for “off-label” presentations including, spasmodic dysphonia,
oromandibular dystonia, truncal dystonia, limb dystonia, and tardive dystonia.
Toxins 2024,16, 261 9 of 29
Table 2. Different formulations of BoNT-based therapies.
Abo-
Botulinum-
Toxin A
(Dysport®)
Inco-
Botulinum-
Toxin A
(Xeomin®)
Ona-
Botulinum-
Toxin A
(Botox®)
Rima-
Botulinum-
Toxin B
(Myobloc®/
Neurobloc®)
Pra-
Botulinum-
Toxin A
(Nabota®)
Leti-
Botulinum
Toxin A
(Botulax®)
Daxibotulinum-
Toxin A (Daxxify®)
Toxinotype A1 A1 A1 B A1 A1 A1
Strain Hall Hall Hall Bean Hall CBFC26 Hall
Complex
Size >500 kD 150 kD 900 kD 700 kD 900 kD 900 kD 150 kD
Excipients
HSA (125
µ
g)
Lactose
HSA (1 mg)
Sucrose
HSA (500 µg)
Sodium
chloride
HSA
(500 µg/mL)
Sodium
succinate
Sodium
chloride
HSA
(500 µg/mL)
Sodium
Chloride
0.9 mg
HSA
(250 µg)
Sodium
chloride
0.9 mg
RTP004 peptide
11.7 µg, L-histidine
(0.14 mg),
L-histidine-HCl
monohydrate
(0.65 mg), polysorbate
20 (0.1 mg), Trehalose
dihydrate
(36 mg).
Solubilization
Saline
solution
Saline
solution
Saline
solution N/A Saline
solution
Saline
solution
pH 7 7 7 5.6 5.5
Units/vial 300, 500 100, 200 100, 200 2500, 5000,
10,000 100 100, 200 50, 100
Shelf life
(Months) 24 36 36 24 36 24
Protein
content
(ng/vial)
4.35 0.6 5 25, 50, 100
Adapted from [71,72,76].
Table 3. Some recent BoNT formulations in clinical trials.
Name
Other Name
Toxinotype Indications Status Origin
Prabotulinumtoxin A ABP-450 BoNT/A1 Migraine, cervical
distonia Phase 2 AEON Biopharma,
Irvine, CA, USA
Neubotulinumtoxin A none BoNT/A1
Primary axillary
hyperhidrosis,
cervical distonia
Phase 3 Medytox, Inc., Seoul,
Republic of Korea
Letibotulinumtoxin A none BoNT/A1 Glabellar Lines Phase 3 Hugel Inc., Seoul,
Republic of Korea
Relabotulinumtoxin A QM-1114 BoNT/A1 Glabellar lines and
lateral canthal lines Phase 3 Galderma, Courbevoie,
France
Nivobotulinumtoxin A Innotox BoNT/A1 Glabellar Lines Approved in
Korea
Medytox, Inc., Seoul,
Republic of Korea
Trenibotulinumtoxin E none BoNT/E Glabellar Lines Phase 3 Allergan Aesthetics,
Irvine, CA, USA
A2NTX none BoNT/A2 Cervical distonia Phase 1
Shionogi Pharma, Osaka,
Osaka, Japan
IPN10200 none Engineered A/B
Adult upper limb
spasticity; upper
facial lines
Phase 2 Ipsen Pharma,
Paris, France
Gemibotulinumtoxin A none BoNT/A1 Post-operative atrial
fibrillation Phase 2 AbbVie, North Chicago,
IL, USA
Daxibotulinumtoxin A none BoNT/A1 Adult upper limb
spasticity; migraine Phase 2 Revance, Nashville,
TN, USA
Toxins 2024,16, 261 10 of 29
3. An Expanding Range of Indications
In addition to the conventional indications as described above, BoNT-derived formula-
tions are benefiting a much wider range of patients suffering from diverse disorders. Some
of the “off-label” applications are usually approved by the regulators under emergency
indication protocols, while the neurogenic bladder indication is already fully approved by
the FDA and EMEA.
3.1. Urology
Over the last 20 years, ONA and ABO have been extensively studied on Urological
pathologies, first in patients suffering from various neurological disorders leading to
neurogenic detrusor overactivity, then later in overactive bladder, and finally in painful
bladder syndrome/interstitial cystitis (PBS/IC) patients.
Initially, the rationale for using botulinum toxin in urology practice was based on their
strong inhibition on motor effects, through blockade of cholinergic neurotransmission in
the detrusor. However, a huge number of preclinical studies in rodents suggested that the
positive effects in reducing bladder contractility and micturition reflex could be mainly due
to an effect on sensory pathways, through the inhibition of some neurotransmitters (i.e.,
nitric oxide, substance P, and CGRP) from the afferent nerve terminals. This hypothesis is
strongly supported by the fact that antimuscarinics have limited efficacy in the majority of
patients suffering from urinary bladder dysfunctions. However, most preclinical studies
remain to be interpreted with caution considering the high doses used to observe positive
effects on bladder contractility in vivo [77].
3.1.1. Neurogenic Detrusor Overactivity (NDO)
Currently, only ONA is approved for the treatment of NDO [
69
]. NDO could be a
consequence of neurological diseases like stroke, spinal cord injury (SCI), and Parkinson’s
Disease, in addition to multiple sclerosis or transverse myelitis. NDO is characterised by
several dysfunctions of the autonomic control of the urinary bladder and urethra because
the signalling between the CNS and lower urinary tract is impaired. Urethral-sphincter
dyssynergia induces a great increase in urinary bladder pressures and can lead to vesico-
ureteral reflux and hydronephrosis.
Recently, the results of two phase III randomised studies (CONTENT1 and CON-
TENT2) in patients with NDO treated with intra-detrusor injections of ABO 600 U or 800 U
vs. placebo were published. ABO was well tolerated and significantly improved continence
and bladder function, and QoL, in patients with SCI or MS with NDO [
78
]. These results
could lead, in the near future, to the approval of ABO for NDO patients.
A randomised, double-blind, non-inferiority clinical study was performed on the effi-
cacy and tolerability of INCO vs. ONA intra-detrusor injections in patients with refractory
NDO performing intermittent catheterization. The authors concluded that INCO was not
inferior to ONA in improving clinical and urodynamic findings in the short-term follow-up,
with comparable adverse effects [79].
Resistance to BTX-A is a significant problem in patients with NDO. A loss of efficacy
over time has been described using ex vivo bladder model highlighting the need for a better
understanding of the underlying mechanisms of BoNT actions [
80
]. Moreover, in a very
limited study in four patients, INCO (which is free from complexing proteins) showed a
good therapeutic response in one out of four patients, the only one not resistant to ONA in
the extensor digitorum brevis (EBD) test [
81
]. The authors suggested that resistance could
depend on antibodies working against the complex protein of ABO and ONA rather than
from antibodies against the neurotoxin itself, as previously hypothesised. Nevertheless,
the binding of antibodies to the accessory proteins does not automatically translate into the
inhibition of the toxin’s effects, and the presence of antibodies does not automatically impact
upon patient non-response to treatment [
82
]. Rather than a specific antibody response,
the physiology of compensatory mechanisms should be further explored to explain the
resistance to BoNT injections in NDO patients.
Toxins 2024,16, 261 11 of 29
3.1.2. Overactive Bladder
Today, only ONA is approved by FDA for the treatment of OAB. This condition affects
a large percentage (between 10 and 20%) of the general population, and almost a third of
these patients are affected by urinary incontinence, which severely impacts their quality
of life. Anticholinergics are the most used pharmacological class of oral drugs, but their
efficacy is limited and since they have numerous side effects and very high discontinuation
rates are observed worldwide.
In the first large phase 3, placebo-controlled trial (EMBARK Study) of ONA in patients
with overactive bladder and urinary incontinence inadequately managed with anticholin-
ergics, ONA (100 U) significantly decreased the daily frequency of urinary incontinence
episodes vs. the placebo. Moreover, 23% vs. 6% of patients became completely continent [
83
].
Urinary tract infection was the most common adverse event, followed by urinary retention.
These results were confirmed by a study on Japanese patients, inadequately managed
with anticholinergics and/or
β
3-adrenergic receptor agonists and injected with one dose of
ONA at 100 U [84].
3.2. Off-Label Uses
3.2.1. Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS)
This pathology is more common than previously reported [
85
]. The aetiology is
unknown, but curiously, 90% of patients are women. Ulcer-type and non-ulcer-type
IC/BPS are considered different disease entities. In a clinical study, it was found that
repeated intravesical ONA injections provided some benefits in half of the patients with
non-ulcer-type IC/BPS, but did not have any effect on patients with more severe disease,
like ulcer-type IC/BPS [86].
In a recent phase II study on women, ONA 100 U was administered as 10 trigonal
injections. The proportion of patients who achieved a 50% or greater reduction in the
pain visual analogue scale was 60% for ONA vs. 22% for placebo, suggesting a significant
therapeutic effect and good safety [87].
In conclusion, ONA is very effective in treating NDO and OAB at a dose of 100 U,
since doses at 200 or 300 U are associated with more frequent side effect, mainly urinary
tract infections and urinary retention, requiring urinary bladder catheterization.
However, there is still a need to increase the efficacy and duration of the therapeutic
effects of botulinum toxin in patients suffering from NDO, OAB, and IC/BPS and, in the
meantime, to reduce side effects. Moreover, since a significant drawback is represented
by toxin administration, i.e., detrusor injections performed at hospital under anaesthesia,
some companies are developing new formulations of botulinum toxins to be administered
through a catheter as an intravesical solution [88].
3.2.2. Female Sexual Dysfunctions
There is growing evidence suggesting that BoNT is a safe and efficacious treatment
option for female patients suffering from various sexual and genitourinary disorders, like
dyspareunia, vaginismus, vestibulodynia, and persistent genital arousal disorder. In those
patients, the BoNT is deemed to act by inhibiting the release of inflammatory neuropeptides
CGRP and substance P and by inhibiting the acetylcholine release at the neuromuscular
junction leading to reduced resting muscle tone. However, extensive research is still
required to precisely identify the exact mechanisms in such disorders and to optimize the
dose injected as well as the injection techniques [
89
]. In conclusion, more randomised
controlled trial data regarding its long-term safety and efficacy are necessary to support
these indications [90].
3.2.3. Erectile Dysfunctions
The first indication of the beneficial effects of BoNT in erectile dysfunctions (ED)
were evidenced during human and animal studies conducted by two different groups of
investigators showing pro-erectile effects by an intra-cavernous injection of BoNT-A. The
Toxins 2024,16, 261 12 of 29
pro-erectile function could be due to cavernous smooth muscle relaxation by inhibition
of noradrenaline release from the adrenergic neurons acting on the cavernous smooth
muscle. In a clinical trial, an increase of 5 to 10% in erectile score in patients receiving BoNT
was described [
91
,
92
]. Guliano et al. evidenced the role of sympathetic hyperactivity in
ED which, therefore, can benefit from BoNT injections [
93
–
96
]. The erection mechanism
implies a complex and delicate co-ordinated equilibrium between neurological, vascular
and tissular compartments. There is a need for randomised placebo-controlled trials to
further investigate the complex mechanisms leading to improved ED, since reliable data
about sympathetic overactivity are missing to date. In addition, the current intracavernosal
injection method will require further optimisations to improve patient comfort. Neverthe-
less, neither prolonged erection nor priapism were observed during the aforementioned
clinical studies, in line with a reassuring locoregional safety profile and appropriate dosage
of BoNT injections.
3.2.4. Acute Dysmenorrhoea and Pelvic Pain Syndrome
Severe forms of dysmenorrhoea represent a common complaint in women and with
major impact on their quality of life, fertility, productivity, and healthcare utilisation. Severe
dysmenorrhea-limiting activity occurs in 16–29% of women and 5–7% have acute dysmenor-
rhoea [
97
]. The pelvic pain syndrome from uterine origin (painful uterine syndrome: PUS)
is very frequently associated with severe dysmenorrhea syndrome along with culpability
and hysterisation. Several magnetic resonance studies (cine-MRI) confirmed the significant
increase in uterine myometrial hypercontractility in dysmenorrhoeic patients [
98
]. The
PUS is the result of hypersensitivity and hypercontractility involving the pain regulation
with exacerbated nociception involving substance P and leading to a decreased sensitivity
threshold. Such sensibilisation mechanisms suggests considering the use of Botulinum
Toxin (BTX) injections for treating acute dysmenorrhea and PUS. BTX injections have previ-
ously been recognised to be effective in patients with an overactive bladder, while BTX’s
effectiveness is somewhat less evident in PUS. Nevertheless, its efficiency in PUS has been
clearly confirmed in randomised controlled studies or meta-analyses [99–101].
3.2.5. Chronic Pain
BoNTs are classically described as inhibiting the neurotransmitter release through
exocytosis blockade. However, neuronal hyperexcitability driven by ionic currents has
also been shown to be blocked by BoNT, demonstrating that BoNT actions are not limited
to SNARE proteins cleavage [
102
]. The injection of BoNTs produced the inhibition of
neurogenic inflammation in different pathologies like phantom pain and neuropathic
neuralgia. In their clinical study of BOTNEP, Ranoux and coworkers established the
beneficial effect of intradermal injection of BoNT in allodynia by improving the pain
threshold [
103
,
104
]. However, the experimental data remain difficult to translate from
healthy volunteers exposed to acute pain stimuli to sustained pathological pain since pain
is greater in chronic pathological pain. Moreover, a possible central action of BoNT/A
cannot be excluded since relief is obtained in the long term after one single injection.
The elucidation of the mechanism of action is still required in the effects of BoNT/A
in neuropathic pain. In addition, the topical application of BoNTs is considered in the
elderly patients to avoid iatrogenia. In the future, toxin subtypes that would selectively
target nociceptives fibres as well as cutaneous delivery techniques should be considered to
improve the safety profile of BoNTs.
3.2.6. Trigeminal Neuralgia
The application of BoNT in trigeminal neuralgia was evidenced by Zhang and cowork-
ers using intradermal injections [
105
]. Nevertheless, considering the local side effects,
experimental studies on new delivery formulations are required to optimise this indication
since BoNT injections are also performed at specific trigger-points [
104
]. The clinical efficacy
Toxins 2024,16, 261 13 of 29
of BTX-A in trigeminal neuralgia is due to the inhibition of the release of inflammatory
mediators and peripheral neurotransmitters from sensory nerves [106,107].
3.2.7. Low Back Pain, Sciatica, and Pyalgia
BoNT injections are beneficial in lombalgia, pyalgia, myofascial pain, and sciatica.
Stretching sessions are still useful before BoNT injections as well as massage of the painful
site. Clinicians face the challenges of complicated diagnosis and lack of knowledge of BoNT
pharmacology. However, a recommended consensus dosage of 100 units divided in 2 to
3 muscle points was agreed [
108
]. Although BoNT efficiency is recognised, several questions
remain to clarify for this indication such as the localisation and dose regimen optimisation.
3.2.8. Arthropathy
The intra-articular administration of BoNT/A for pain relief in musculoskeletal disor-
ders has stirred great interest among patients suffering from long-term chronic pain. While
BoNT was historically used in humans to reduce muscle spasticity, recent preclinical studies
show an intrinsic intra-articular antinociceptive effect during BoNT treatment [
109
–
111
].
The pharmacodynamics behind pain modulation remain unclear since pain in arthropathy
involves both nociceptive, neuropathic complex mechanisms, and abnormal excitability in
peripheral and central pain pathways. BoNT is deemed to act as a “Swiss knife” by locally
suppressing the neurotransmitter release leading to reduced peripheral sensitisation as well
as decreased central sensitisation [
112
]. BoNT has an inhibitory role on the release of critical
neuromediators involved in nociception, including substance P, calcitonin gene-related
peptide, and glutamate.
Arthropathy is usually treated with analgesics, corticoids, and anti-inflammatory
drugs as well as hyaluronic acid; however, these drugs show a modest effect along with
side effects. In this indication, the action of BoNTs can complete the therapeutic arsenal.
To date, 14 randomised clinical trials have confirmed the analgesic effects of BoNT in this
indication. The expected side effect consisting of possible reduced motor activity and
lasting almost 8 weeks has been accepted by patients who all regularly return to the clinic
to receive a new dose of BoNT because of its efficacy [113,114].
3.2.9. Benefits of BoNT Injections in Cancer Therapies
More recently, many studies have raised awareness on the benefit of BoNT injections in
painful symptoms associated with cancer and resulting from the direct pressure generated
by the tumour or from chronic neuropathic pain after surgery or radiation. Nine studies
among 10 clinical studies using a standardised scale to measure pain (VAS) demonstrated
statistically significant improvement of the local pain at 4–8 weeks post-BoNT injection
compared to baseline [
115
,
116
]. The use of BoNT injections can help to relieve pain in
patients suffering from post radiation pain, hyperalgesia, and peripheral neuropathy after
chemotherapy or postsurgical pain. For example, one single injection of BoNT can be
sufficient in pectoralis muscles to avoid pain after a mastectomy. In the case of head and
neck surgery, most clinicians agree that pain improves after injection of BoNT [117].
Patients with neuropathic pain due to spasm after a laryngectomy respond very well
to 20 UI of Onabotulinum A for 10 days. Gastroparesis after oesophageal surgery is also
greatly improved by 100 Units of Onabotulinum A. Nevertheless, more studies are still
required to improve efficiency and safety of such injections. In addition to significant relief
of pain, Onabotulinum A and Rimabotulinum B significantly reduce intense sweating of
the face or excessive salivation post-surgery. The cessation of the hyperhydrosis due to
parotid gland surgery, after BoNT injections is highly promising for the patient quality of
life [
118
,
119
]. Parotid fistula healed rapidly after BoNT injections, while no serious adverse
events were observed [
120
]. Chemotherapy-induced Raynaud syndrome is also improved
after injection of BoNT in the hand. Tingling pain, spasm of the hand, and spasm of small
arterioles are reduced, which can avoid ulcer and gangrene risk [121].
Toxins 2024,16, 261 14 of 29
In parallel to clinical benefit in cancer patients, experimental studies show that BoNT
exposure is efficient against cancer cells proliferation in culture and
in vivo
. Cellular
apoptosis and the reduction in tumour size were evidenced after injections of BoNT into
malignant tumour models associated with antiproliferative effects and increased caspase
3/7 activity [
122
–
124
]. Yet, the BoNT dose and exposure durations used in experimental
studies are not directly transferable to the dosing regimen applied in human patients.
Progress is being made to deliver BoNT to cells selectively (breast cancer cells
in vitro
) since
early data suggest that the BoNT/A apoptotic activity may be selective for cancer cells, but
additional studies are still required to assess the translational value of experimental
in vivo
or in vitro findings to human patients [125].
To summarise clinical and experimental findings, there is serious suggestion that
BoNT can help cancer patients suffering from acute or chronic pain. Since the very origin
of the pain comes from the muscle, from the neuropathic injury or the inflammation due to
peripheral nerve entrapment, BoNT can bring an incredible benefit to the patient acting
on each pathophysiological process. Botulinum toxin treatment offers two major benefits
over classical pain remedies: BoNT injection effects last from 3 to 6 months and BoNTs
are localised and safer than all potent analgesic agents. All the clinical studies performed
recently in cancer patients after a surgery or a radiation therapy confirm the efficacy and
safety of BoNTs on the pain associated with surgical and radiation therapies. BoNT is also
very helpful in orofacial pain induced by chemotherapy.
Nevertheless, safety studies are required to guarantee safe dosing. Indeed, last year, a
botulism outbreak occurred in Turkey following the injection of excessive BoNT doses in
the stomach of patients [
126
]. Nevertheless, the potential benefit of BoNT is very promising
because of the increasing incidence of cancer.
Finally, it is worth noting that only two BoNT toxinotypes are currently approved
for clinical use (BoNT/A and BoNT/B) despite the existence of at least eight different
toxinotypes, BoNT A to H (or mosaic FA), thus suggesting a wider therapeutic potential [
29
].
Considering the increasing interest in therapeutic applications of BoNTs, it is becoming
very attractive to enhance the properties of BoNTs by modulating its potency, eliminating
its immunogenicity, extending the effects duration, developing fast acting formulations, or
retargeting the toxin towards sensory neurons. Extending the duration of action of BoNT
would benefit patients suffering from chronic conditions like muscle spasticity, overactive
bladder or chronic migraine. The fast-acting formulations would be beneficial to deal with
acute pain and also to expand the cosmetic uses.
4. Exploration of Therapeutic Potential of BoNTs Toxinotypes or Subtypes
The rich diversity of BoNTs toxinotypes or subtypes serves as the foundation of new
therapeutics. In addition, the considerable progress in sequencing is shedding light on the
expanding BoNT superfamily as a natural repertoire available to support novel therapeutic
options and the design of tailored toxins.
4.1. Variability between Toxinotypes
The pioneering studies on the structure of BoNTs by Lacy et al. described their moving
modular shape “bringing the toxin molecule to life”. It became possible to grasp the
correlation between structural particularities of each BoNT toxinotype and their functional
diversity [
127
]. A key difference in subdomain organisation was evidenced in BoNT/E
by Kumaran et al. in 2008, while BoNT/A and BoNT/B share a high degree of similarity.
The catalytic and binding domains of the BoNT/E are arranged on the same side, which
correlates well with the more rapid onset of action of BoNT/E. Those structural properties
highlight that BoNTs toxinotypes do not share the same modular special organisation
which greatly influences their effects [128,129].
In line with those differential effects and based on animal studies, the BoNT/B tox-
inotype was found to be less potent than anticipated in the clinic. The lower efficiency in
humans was recently identified to be related to a residue difference within human synapto-
Toxins 2024,16, 261 15 of 29
tagmin II (protein receptor for BoNT/B) [
130
]. This residue lies within the synaptotagmin
II-binding cleft causing a lower affinity for BoNT/B than in other species. As a result,
higher doses of BoNT/B need to be injected to achieve a similar efficacy than that achieved
with BoNT/A [
131
]. This finding has prompted the design of optimised BoNT/B sequences
binding to human synaptotagmin II with higher affinity and leading to enhanced efficacy.
Although modified BoNT/B proteins show promising potential in the human neurons
derived from induced pluripotent stem cells and transgenic humanised mouse models, the
target doses need to match available BoNT/A1 products in order to claim a comparable
efficacy and safety profile. The 4-fold difference in EC50 observed between rBoNT/A1
and rBoNT/B1 in the humanised mice contrasts with the 40-fold conversion factor used in
the clinic between BoNT/A1 and BoNT/B1, but it still implies that the lower affinity of
BoNT/B1 is not the only reason for its lower clinical efficacy. There are also large differences
in the expression of human syt1 and syt2 between the different cell models that suggest
only partial transferability to human tissues [132].
BoNT/A, BoNT/B, and BoNT/E cause neuromuscular paralysis for more than 4 months,
~2 months, or
≤
4 weeks, respectively, when applied locally for the treatment of dystonia,
illustrating the differences in neurotransmitter release blockade periods between differ-
ent BoNT toxinotypes. The inhibition of transmitter release from cerebellar neurons by
BoNT/A is far longer than with BoNT/B. Similarly, the BoNT/E and BoNT/F cause the
short-lived blockade of transmitter release that coincides with the reappearance of intact
SNAREs confirming their short half-lives of inhibition. The therapeutic potential of BoNT
toxinotypes A1 to F1 were evaluated in ex vivo,
in vitro
, and
in vivo
assays. Except for
toxinotype D1, all BoNT toxinotypes were evidenced as highly potent neurotoxins in ro-
dent assays,
in vivo
. However, some intriguing dissimilarities between peripheral and
central neurons suggest that further investigation is needed to clarify differences between
toxinotypes in humans depending on the injection localization [133,134].
It is anticipated that the specificities of each BoNT toxinotype can lead to unique
BoNT-based therapeutic formulations. In particular, toxinotypes F1 and C1 represent
candidate therapies aimed at somatosensory system modulation [
67
]. The rapid refilling
of synaptobrevin or SNAP-25 pools inside the cells explain the shorter effects of BoNT/F
and BoNT/E, whereas the longevity of their respective proteases and cleavage products
support the prolonged effects of BoNT/A, BoNT/B, and BoNT/C1 [
133
]. In addition, it was
shown that the BoNT/A1 light chain’s longer lifetime is due to its ability to escape the cell
degradation systems such as ubiquitination [
134
]. Conversely, the BoNT/E light chain is
indeed ubiquitinated and driven towards the ubiquitin–proteasome system. This resistance
to ubiquitination is due to the ability of BoNT/A light chain to recruit deubiquitinases that
destroy the polyubiquitin chains acting within the ubiquitin–proteasome system. These
differential durations of action can support the design of new BoNT formulations with
differential pharmacokinetic profiles to fit different therapeutic needs [
129
–
136
]. Never-
theless, the differences in specific potency need to be interpreted with caution since they
may be related to differences in toxin purity, toxin manufacture, or experimental conditions.
Future, more detailed clinical studies on the different serotypes would help the elucidation
of their pharmacokinetic profiles.
Assessing the differences in duration of action is paramount in clinical settings. This
diversity between different BoNT toxinotypes provides to clinicians a unique opportunity
to adapt the efficacy, duration of action, and antigenic potential of each BoNT formulation.
BoNT/F was found to induce earlier sprouting and complete recovery faster than with
classical formulations containing BoNT/A or BoNT/B. Electrophysiological studies can
support physicians’ choices regarding BoNT formulations based on data obtained for each
patient. The choices between different BoNT serotypes for the clinicians results from the
analysis of preclinical and clinical studies, carefully assessing the relative efficacy, duration
of action, safety, and antigenic potential of each serotype [
136
]. A new BoNT/A-B hybrid
was designed, combining the high potency of BoNT/A with the high specificity of BoNT/B.
Interestingly, in this study, the BoNT/A-B hybrid showed the same potency and duration
Toxins 2024,16, 261 16 of 29
than with BoNT/A-induced paresis
in vivo
, while the potency of the hybrid BoNT was
8-fold higher in the ex vivo hemidiaphragm assay. This combination will facilitate a dose
reduction as well as a prolongation of the time interval between two administrations in
autonomic disorders; however, differences between
in vivo
and ex vivo data show that the
binding kinetics of BoNT/A-B hybrids differ between experimental settings [137].
In addition to modulation of the pharmacokinetic profile, the use of diverse toxino-
types can support new indications. Humans are not responsive to BoNT/DC targeting
VAMP-2; however, the mosaic toxin BoNT-DC, but not BoNT-A, reduced melanin content
in multiple melanocyte models, probably due to the cleavage of VAMP leading to the
aberrant trafficking of tyrosinase and other cargo required for melanogenesis [138].
4.2. Variability between BoNT Subtypes
The duration of the effects of BoNT/A subtypes 1 to 5 was assessed in primary rat
spinal neurons considering that the effects can reach 2–6 months in patients, in particular
for BoNT/A. In this study, the intracellular activity of BoNT/A1, /A2, /A4, and /A5
could last for 10 months, whereas BoNT/A3 effects only lasted for 5 months. In the case of
BoNT/E, the intracellular effect only lasted for 2–3 weeks. Those experimental findings
demonstrate that the differential longevity of BoNT/A subtypes can be exploited to design
long-lasting formulations [139].
The same authors revealed marked differences between the ten BoNT/A subtypes
identified to date, consisting of differences in cell entry or proteases dynamics to differences
in toxin potency
in vivo
and
in vitro
in the neuronal cell model. They confirmed that
injections of BoNT/A1, /A2, and /A6 induced similar levels of paralysis as measured
in the digital abduction paralysis model. Similarly, injections of BoNTs A1, A2, A6, and
B1 resulted in a comparable pattern of paralysis using the Catwalk set-up. However,
the effects of BoNT/B1 occurred one day later than with other BoNT/As [
139
]. The
authors previously confirmed the comparable long-lasting effects of BoNT/A1, BoNT/A2,
BoNT/A4, and BoNT/A5 subtypes in cultured primary neurons, while the BoNT/A3 effect
was significantly shorter. In addition, a faster onset of paralysis was observed with a local
injection of BoNT/A2
in vivo
than with BoNT/A1, BoNT/A3, BoNT/A4, and BoNT/A5,
as shown previously by different authors, while a faster recovery in motoneurons was
observed with BoNT/A3. Currently, BoNT/A2 is being investigated in clinical trials in Japan,
and A6 was also suggested as a potential new pharmaceutical. A clear benefit of the Catwalk
model is the possible evaluation of the effects of BoNTs in a wide range of conditions like
arthritis, spinal cord injury, Parkinson’s disease, multiple sclerosis, and stroke [140].
However, regarding BoNT variability, the functional properties of the various subtypes
remain to be explored in additional
in vitro
or
in vivo
models. BoNT/A2 showed a faster
onset than BoNT/A1 and a significantly higher potency in the
in vivo
and ex vivo models. In
the same way, significant differences were evidenced between the paralytic effects of BoNT/B1
and BoNT/B2 ex vivo and
in vivo
[
141
,
142
]. Conversely, while causing different symptoms
of intoxication in mice than with BoNT/A1, BoNT/A3 was found to be less potent and
less effectively neutralised by anti-BoNT/A1 antibodies. These differences were suggested
to originate from significant structural differences within the BoNT/A3 binding domain
(Hc/A3) [
143
]. Likewise, the different mutations discovered in the BoNT/A3 and A4 binding
domains may influence their binding dynamics and the different symptoms observed [144].
In addition to those findings, Tian and coworkers evidenced that while the nucleotide
variability is uniformly distributed along the BoNT gene, the amino acid variability is
not uniform within the full protein. The amino acid differences are focused along the
light chain (LC) subdomain and the C-terminus of the receptor binding domain (HCC).
Consequently, the LC region and the HCC region of BoNT are deemed to be the main
source of BoNT differentiation. This finding explains the BoNT ability to bind to different
receptors and cleave different substrates, thus targeting a wide variety of hosts [
145
]. Since
the LC controls the catalytic properties and the duration of BoNT action, variations in the
Toxins 2024,16, 261 17 of 29
LC will directly impact the therapeutic properties of BoNT. Understanding such properties
of the LC will support targeted applications of BoNT in human therapies [26].
This variability was confirmed in the study by Johnson and coworkers, who analysed
two newly sequenced BoNT/A variants, Loch Maree (A3) and 657 Ba (A4), in comparison
to A1 and A2. Combining sequence analysis, functional effects, molecular modelling and
crystal structures of BoNT/A1 and the light chain of BoNT/A2, they concluded that the se-
quence differences within subtypes directly impact the isolation of efficient broad-spectrum
antibody and small inhibitors. The significant differences between BoNT/A3 and BoNT/A4
in binding affinity and cleavage efficiency particularly affects their S1’ subsite recognition [
146
].
Further functional and modelling studies indicated that in rodent models, the disruption of
HCc-V2C
β
-peptide and -glycan-N559 interactions mediates low BoNT/A4 potency, while in
human motor neurons, the disruption of HCc-SV2C
β
-peptide alone mediates low BoNT/A4
potency due to species-specific variation at the SV2C site [147].
More recently, the progress made in the understanding of the lipid binding loop–
interactions has supported the design of a more potent BoNT/B mutant. The mutant
BoNT/B combines a stronger potency with lower blood diffusion than the original native
BoNT/B. Therefore, this BoNT/B mutant combines higher safety with enhanced efficacy [
148
].
Considering the considerable progress made in BoNT variable structure elucidation, it
becomes essential to translate recent structural findings into the design of more efficient
and more specific therapies based on BoNTs protein models.
5. Modelling BoNTs for Non-Neuronal Targeting
While being one of the deadliest substances on earth, BoNT is used as a therapy in a
variety of applications including dystonia or pain due to its exclusive ability to selectively
target neuronal membranes. Using molecular engineering, this aptitude to select specific
territories can now be exploited to target non-neuronal tissues while minimising systemic
exposure [
149
]. Moreover, this precise targeting ability is essential to explore neuronal
physiology or design restorative therapies.
The initial approach by Dolly et al. consisted of engineering native BoNTs into inac-
tive mutants (full-length BoNTs incorporating catalytically inactive LC/A or BoTIMs) and
combining the inactive mutants with native LC/E domains. The resulting mosaic protein
combined the persistence of LC/A with the fast onset of the LC/E action. This persistent
cleavage of SNAP-25 may benefit patients with chronic pain syndromes or
migraine [150,151]
.
In addition, the LC/E is more potent in the inhibition of CGRP release from sensory neurons,
while the BoNT/A is more specifically targeting sensitive neurons, which confirms the benefit
of combining the unique properties of each toxin component [152,153].
Studies were carried out to enhance the effect of LC/B on VAMP cleavage
in vitro
;
however, the resulting BoNT/B mutant did exhibit a higher efficacy in
in vitro
assays (cell-
based assays) or
in vivo
models [
154
]. The role of BoNT/C as a potential therapeutic toxin
for synaptic transmission modulation was also evidenced by specific mutations enhancing
the cleavage of syntaxin-1 [
155
,
156
]. Later, Elliott et al. isolated a newly engineered
botulinum neurotoxin B combining improved binding ability with enhanced potency in
preclinical models [132].
Compared to the modulation of the binding properties, the modulation of the translo-
cation process remains challenging since the translocation dynamics remain a matter of
debate. Mutated proteins conserve negatively charged residues in the LC and HN of
BoNT/B on the basis that the protonation of these residues is required for the interaction of
the toxin with the negatively charged membranes. Pirazzini and collaborators produced
a triple mutant with a higher potency and faster onset time due to the protonation of
the protein residues involved in the translocation of BoNTs. Nevertheless, BoNT protein
engineering based on the translocation process remains challenging [157].
Their modular structure–function relationship makes BoNTs proteins more amenable
candidates for retargeting to different territories. The fragment of BoNT/A composed of
the LC and HN domains (LHN) unable to bind to neuronal cells has less toxicity, but the
Toxins 2024,16, 261 18 of 29
remanence of the translocation domain can still facilitate the formation of pores across
the membrane under acidic conditions [
158
]. The crystal structure of LHN/A revealed a
preserved structural relationship despite the absence of the HC domain, which makes the
LHN fragment a common tool to deliver LC into cells that are not usually targeted by BoNTs
in the nature [
48
,
159
]. The first studies employing this approach involved the use of an
LHN/A preparation chemically coupled to lectin wheat germ agglutinin and nerve growth
factor (NGF). The resulting conjugate was able to inhibit the release of noradrenaline from
PC12 cells showing that the endopeptidase can be successfully delivered into a range of
neuronal and non-neuronal cell types [160].
Based on the exclusive properties of the BoNTs, a new class of active proteins was created
(TSIs: targeted secretion inhibitors; or TVEMP: targeted vesicular exocytosis-modulating
protein) comprising three basic domains: (1) the LC domain of one selected BoNT toxinotype
having the ability to cleave the SNARE proteins depending on the BoNT toxinotype, (2) the
HN domain providing the intracellular translocation ability of the LC, and (3) the binding
domain derived from BoNT or from a peptide or a protein interacting with a specific receptor
on the target cell. This new approach exploits the endopeptidase domain to modulate the
intracellular processes of the target cells by inhibiting their secretion mechanisms [5].
Figure 1summarises the BoNT engineering opportunities.
The recombinant expression and purification of the LHN fragment of BoNT from
E. coli allowed the combination of the LHN with a targeting entity. This technique can
extend the cleavage activity to a variety of SNARE proteins opening new opportunities
to target non-neuronal cell types that are not expressing SNAP-25 constitutively [
161
]. As
an example, it is now possible to design new BoNTs to treat disorders related to SNAP-
23-mediated hypersecretion based on the co-crystal structure, molecular dynamics, and
mutagenesis findings. BoNT-derived fusion proteins are able to specifically target cell types
involved in secretion mechanisms providing a basis to formulate new secretion inhibitors.
A similar approach was used with the coupling of LHN/C to epidermal growth factor
(LHN/C-EGF) to inhibit the release of mucin by pulmonary epithelial A549 cells in the
treatment of asthma or chronic obstructive pulmonary disease [162].
The progress in understanding the structure–function relationship of BoNTs has
permitted to overcome the SNAP-25 substrate limitations and extend the area where BoNTs
may exercise its action [
163
–
165
]. The extension of BoNT substrate specificity has made
possible to block the release of interleukin-8 (IL-8) and mucin from stimulated HeLa cell.
This opportunity for a mutated LC part of BoNT/E to impact vesicle trafficking within
non-neuronal tissue significantly expands the therapeutic applications of the BoNTs [
166
].
Another approach employs a protein-stapling technology to re-assemble BoNT/A
from two separate fragments to generate a uniquely safe tool for neuroscience studies
and new therapeutic applications. This BoNT-derived stapled protein chimera can reduce
the mechanical hypersensitivity observed in a rat model of inflammatory pain and blocks
the visual cortex neuronal activity but without inducing paralysis. Thus, this protein
stapling technology allows the assembly of distinct BoNT fragments to yield new molecular
properties without side effects [164].
Conversely, BoNT engineering may be used for the delivery of cargo or active com-
pounds directly into neurons. Typically, the targeting of specific cells by biological toxins
involves the delivery of their enzymatic moieties into the cytosol, but this delivery mode
can be exploited through protein engineering to produce chimeric toxins. The Clostridium
botulinum C2 binding and translocation domain was retargeted to neural cell populations
by replacing its binding domain with a BoNT binding domain [
167
]. By using a compa-
rable method, a bioengineered BoNT/C-vehicle was created with the ability to deliver
therapeutic cargo into the neurons [168].
6. Future Approaches and Perspectives
Starting in 1822, when Justinus Kerner envisioned the therapeutic uses of the “fatty
acid agents” that blocked the parasympathetic drive in animals, BoNT was isolated and
Toxins 2024,16, 261 19 of 29
purified at the beginning of the 20th century. Nevertheless, BoNT’s therapeutic poten-
tial was evidenced in strabismus near the end of the 20th century. While the molecular
structure and modular composition of BoNT have been fully elucidated, the number of
therapeutic indications are constantly rising and thus expanding their range from muscle
spasticity, hyperhidrosis, migraine, chronic pain, and cosmetic applications to treatments
for overactive bladder, erectile dysfunction, arthropathy, and cancer.
The engineering of BoNTs was inspired by its modular composition to design more po-
tent, less toxic agents or to retarget the toxin at non-neuronal cells. Nevertheless, additional
studies are required exploiting the engineering of the BoNTs to develop suitable therapies
to treat acute or chronic pain. It is important to note that
in vivo
studies are implemented
in healthy animals, which can be inappropriate in predicting clinical efficacy in spastic
patients since major histopathological alterations are affecting the reactivity of spastic
muscles [
132
]. Currently at the experimental step, the use of engineered BoNTs is becoming
highly desirable for their neuroprotective effects, the influence on neuronal burgeoning,
or on hormone secretion disorders. For example, a CGRP receptor antagonist delivered
BoNT/D protease into sensory neurons and was found to inhibit K+-evoked substance
P release. Since cytokines and neuropeptides are major regulators of inflammation and
pain, blocking their release highlights the bio-therapeutic potential of engineered BoNTs in
numerous chronic diseases [169].
In parallel to molecular engineering, studies on alternative existing toxinotypes evi-
denced the local and long-lasting paralysis effect of BoNT/CD comparable to that induced
by BoNT/A but more efficient than BoNT/C. BoNT/CD was described as a potential
replacement candidate to BoNT/A or BoNT/B in therapy or in cosmetic applications in the
case of patients developing immunity [170].
In addition, a protein stapling technology was developed to combine two identical
binding domains of tetanus and botulinum type D neurotoxins that enhanced intracellular
delivery of molecules into neurons. The duplication of the binding parts of tetanus or
botulinum neurotoxins allowed the production of large therapeutic enzymes penetrat-
ing neurons with higher efficiency [
171
]. The same authors developed a new isopeptide
conjugation system to produce functional botulinum neuronal modulators using the two
non-paralytic parts of the protein. This technique represents a safer approach for manu-
facturing therapeutic botulinum molecules. The elongated el-iBoNT molecule exhibited
reduced paralytic ability compared to the non-elongated version and effectively allevi-
ated nerve injury-induced pain in rats, an important improvement over previous stapled
botulinum molecules [
172
]. Those recombinant forms of BoNTs show the potential to
be targeted to specific neurons and by removing the paralytic moiety of the toxin, this
technique will automatically enhance safety and improve the benefit/risk ratio in therapy.
Regarding the exploitation of differences between toxinotypes, engineering will allow
the improved binding of BoNT/E to SV2c by building a BoNT/A-like binding pocket
within BoNT/E. The modifications of native BoNT/E provide novel avenues for fast acting
BoNT/E-based products [
173
]. A new recombinant BoNT/E has recently been assessed in
a first clinical study showing a faster onset of action, greater peak, and shorter duration of
effect versus abobotulinumtoxinA. This rBoNT-E demonstrated a good safety profile and
will allow unmet therapeutic and aesthetic patient needs to be addressed [
174
]. The faster
onset of action and shorter duration of effect was already confirmed
in vivo
in the mouse
DAS assay; however, this study employed the native form of BoNT/E [175].
The specificity of BoNT/A resides in its high potency and specific binding but with
a lag phase. BoNT/A can now be combined to fast acting inhibitors to gain fast action in
parallel to long-term effects. A combination of conotoxin with BoNT/A has been shown to
accelerate and potentiate the effects of BoNT/A [
176
]. In parallel to faster action, a new tri-
receptor binding BoNT (TAB) has been designed to improve native BoNT pharmacological
profile by optimisation of the binding properties [177].
Toxins 2024,16, 261 21 of 29
Studies on the effects of BoNTs at territories remote from the injection site will support
the development of BoNT therapies specific to certain organs. Currently, the pharmaceutical
development of liquid or slow-release BoNT formulations for transdermal, trans-urothelial,
and transepithelial delivery remains highly challenging. Progress in the formulation and
delivery techniques in parallel to more sensitive analytical techniques will be paramount
to deliver efficient next-generation BoNT clinical products [
48
]. Injection-free delivery
has been tested using liposomal BoNT (lipotoxin). This hydrogel formulation (Theracoat
TC-3) allowed the slow release of BoNT, but its efficacy remained elusive showing that
considerable work is needed to develop efficient “injection-free” formulations [
178
]. In
addition to liposome formulations, microneedle devices (DMNPs) were prepared to test
the functionality of the BoNT/A/DMNPs on the hyperhidrosis mouse footpad, showing
drastically reduced sweat gland activity. Those innovative results demonstrate that these
devices such as microneedles (DMNPs) can be an effective and painless alternative to
hypodermic injections when treating hyperhidrosis with BoNT/A [179,180].
Nevertheless, significant progress has been made regarding the increased duration of
action with the DaxibotulinumtoxinA (Daxxify), a novel BoNTA product containing highly
purified 150 kDa core neurotoxin formulated with a proprietary stabilising excipient peptide
(RTP004) instead of human serum albumin. The positively charged RTP004 enhanced the
binding of the neurotoxin to neuronal surfaces, which facilitates neurotoxin internalisation.
However, its extended duration of action is also provided by the higher dose of BoNT/A
contained in this formulation [
61
]. A new analgesic formulation (N-001) was engineered
from several C. botulinum toxins and targeting sensory neurons resulting in pain relief
lasting for 3 days. Those new results encourage further studies using N-001 as a potential
analgesic for post-operative pain treatment [181].
Another important future aspect will consist in optogenetics approaches allowing the
local activation of the toxin. This formulation of photoactivable BoNTs can be induced to
control locally the neuronal transmission [
182
]. Beyond the activation of BoNTs through
optogenetics, non-replicative viral vectors based on HSV-1 viral particles (Herpes simplex
Virus type 1) can express the LC of BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, and
BoNT/F after infecting embryonic and adult rat DRG neurons. The resulting transgenic
BoNT LCs cleave SNARE proteins, thereby inhibiting the release of CGRP by embryonic
rat sensory neurons. A vector-based strategy could provide a continuous production of
the LC intracellularly, and thus avoid limitations arising from the need to repeat BoNT
administrations and avoid potential resistance to the treatment [183].
To summarise, native BoNT/A or BoNT/B represent widely recognised, effective,
and safe treatments option for a variety of disorders ranging from muscle spasticity and
hyperhidrosis to chronic pain, as well as having had global success in the cosmetic industry.
On the other hand, the optimisation of current formulations is becoming increasingly
sought-after considering the wide potential of the natural BoNT repertoire as well as the
many engineering opportunities lying within this modular protein. Furthermore, new types
of formulations could make BoNT treatments more selective for unique territories while
enhancing their local efficacy and reducing the risk of side effects. However, further clinical
studies are needed to increase the safety margin in the new treatment indications as well
as for the new types of formulations. Despite the high safety profile of present botulinum
toxin formulations, the potential risks related to its intentional and inappropriate uses
need to be thoroughly addressed in view of the expanding formulations and indications.
The existing regulatory framework will require strengthening around the ever-increasing
therapeutic indications. This move towards recombinant and local BoNT formulations
(topic gels, lotions, and microneedle devices) needs to be backed up with comprehensive
safety evaluations both in preclinical and clinical steps. On a positive note, the use of
recombinant biological toxins represents a major advance towards perfect reliability in
toxin production, toxin calibration, and potency quantification. Newly developed reference
materials based on recombinant forms of BoNTs will serve as the basis for the evaluation of
Toxins 2024,16, 261 22 of 29
different detection methods and will be the benchmarks for the assessment of toxin potency
between different laboratories [184].
Author Contributions: Conceptualization, C.R.-E. and S.P.; writing—original draft preparation, C.R.-
E.; writing—review and editing, S.P. and C.R.-E. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article, further inquiries can be directed to the corresponding author.
Conflicts of Interest: The authors declare no conflict of interests.
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