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Arh. farm. 2020; 70: 10 – 19 Pregledni rad/ Review article
Botulinum toxin type A: basic pharmacological
profile and therapeutic applications
Lidija Bach-Rojecky*, Višnja Drinovac Vlah
University of Zagreb - Faculty of Pharmacy and Biochemistry, Department of
Pharmacology, Domagojeva 2, 10 000 Zagreb, Croatia
*Corresponding author: Lidija Bach-Rojecky, e-mail: lbach@pharma.hr
Abstract
Botulinum toxin Type A (BoNT/A), produced by the Gram-positive anaerobic bacteria
Clostridium botulinum, is one of the most potent toxins in nature, and a very useful therapeutic
tool for combating various neurological and autonomic disorders. The main pharmacological
features of BoNT/A are neurospecificity, long-lasting effect, and safety. These features are
grounded on its peculiar molecular mechanism of action: after specific binding to the neuronal
membrane, it is internalized into the neuronal cytosol, where it specifically cleaves one of the
proteins necessary for neurotransmitters release. The consequent reversible neuroparalysis lasts
for several months and explains the long-lasting clinical effects after a single local toxin
application. Although already approved for the prevention of chronic migraine, the basic and
clinical investigations have repeatedly shown the potential of BoNT/A in relieving other chronic
pain conditions. Accumulated data from experimental pain models demonstrated that BoNT/A
reduces pathological pain hypersensitivity after axonal transport to the central nervous system,
where it interferes with complex processes of central sensitization. Future experiments are needed
to explain in more depth BoNT/A molecular mechanism of action and pharmacokinetic
peculiarities.
Key words: botulinum toxin type A, mechanism of action, therapeutic applications,
pain investigation
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Introduction
Clostridium botulinum is an anaerobic spore-forming Gram-positive bacteria which
under appropriate conditions produces different protein neurotoxins (BoNTs). Bacteria
produce seven serotypically distinct BoNTs, denoted as BoNT/A–BoNT/G, which can be
further divided into subtypes according to amino acid sequences. Due to their high
potency and specificity for neurons, BoNTs are one of the most powerful known toxins
in nature (1). Their biological and pharmacological properties have been extensively
studied during the last several decades, with special focus on their potential therapeutic
applications. The therapeutic potential of BoNT/A was unveiled in the 1970s, and
nowadays it has the widest clinical application of all BoNTs.
Structure of BoNT/A
BoNT protein is released from bacteria as a large 900 kDa oligomers consisting of
a 150 kDa neurotoxin and auxiliary proteins, which includes a non-toxic non-
hemagglutinin (NTNH) and three hemagglutinin proteins (2). NTNH contributes to toxin
stability in the acidic environment, while haemagglutinins are involved in translocation
across the intestinal epithelial lining into the lymphatic system and the bloodstream (3).
The neurotoxic part of this progenitor toxin complexes consists of a light chain (L chain;
50 kDa) and a heavy chain (H chain; 100 kDa), which are held together by non-covalent
interactions and a single inter-chain disulfide bond. While the H chain mediates the
specific binding of neurotoxin to the presynaptic plasma membrane of nerve terminals,
the L chain exerts proteolytic activity against specific intracytosolic proteins (3,4).
Molecular mechanism of action
BoNT/A binds with high affinity to peripheral cholinergic nerve terminals and after
entering into the neuronal cytosol blocks the release of acetylcholine from motoneurons
and parasympathetic neurons. The consequence is generalized peripheral flaccid paralysis
of skeletal muscles and dysfunction of cholinergic nervous systems as the most important
pathological features of botulism (4). The mechanism of nerve terminal intoxication by
the BoNT/A includes four steps: 1) specific binding to nerve terminals, 2) internalization
within an endocytic compartment, 3) translocation of the L chain across the vesicle
membrane and its release in the cytosol upon reduction of the interchain disulfide bond;
and 4) cleavage of SNAP-25 (synaptosomal-associated protein 25 kDa), one of the
proteins which is crucial for neurotransmitter release (4). The final result of this specific
series of events is neuroparalysis (Figure 1).
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Figure 1. Molecular mechanism of BoNT/A action on motor nerve terminals
In analogy with its known action on the neuromuscular junction, it was proposed
that BoNT/A prevents the sensory neurotransmitter release from the peripheral nerve
endings as well (5). This was demonstrated with series of in vitro experiments where
BoNT/A inhibited the release of neurotransmitters such as glutamate, noradrenaline,
serotonin, substance P, calcitonin gene-related peptide, adenosine triphosphate,
nicotinamide adenine dinucleotide from cultured cells (4,5).
Clinical applications
BoNT/A has several favorable and unique pharmacological characteristics. As
already described, it is very potent and neurospecific, its action is reversible with time
and finally, it is very safe because of minimal diffusion after local injection in small
volumes. Because of these features, BoNT/A (applied intramuscularly or intradermally
in picogram quantities) is one of the safest and most efficacious therapeutic proteins for
autonomic disorders, spasticity, and hyperkinetic movement disorders, as well as for
cosmetic treatments (6). Since the introduction in human therapy during the 1980s, the
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number of clinical indications has been continuously expanding (Table I). Currently, the
only approved use of BoNT/A concerning pain is chronic migraine, based on randomized
controlled clinical trials (RCTs) and meta-analysis (7).
Table I Therapeutic indications of BoNT/A (Botox) (4)
Neurology focal dystonias (blepharosmasm, cervical dystonia)
non-dystonic disorder (hemifascial spasm)
focal spasticity (limb spasticity)
cerebral palsy
hyperhidrosis
hypersalivation (sialorrhea, drooling in parkinsonian
syndrome)
aesthetics
Ophtalmology
strabismus
Urology
overactive bladder (detrusor o
veractivity)
Pain chronic migraine prevention
Three products that contain BoNT/A are commercially available in this part of the
world: onabotulinumtoxin A (ONA), abobotulinumtoxin A (ABO), and
incobotulinumtoxin A (INCO). ONA and ABO are neurotoxin complexes with auxiliary
proteins (molecular weight 900 kDa) while INCO contains only the purified BoNT/A
(molecular weight 150 kDa). The potency of BoNT preparations is expressed as Units
(U), where 1U corresponds to 1 LD50 in the mouse bioassay (~48 pg). Regarding relative
potency between all three products, in the clinical setting, it was observed that ONA and
ABO have non-parallel dose-response curves, where 1 U of ONA is equivalent o 2.5-3 U
of ABO (conversion ratio is 1:2.5-3), in contrast to ICNO which is equivalently potent as
ONA (8).
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After local injection into the muscle, BoNT/A induces paralysis 2–3 days after the
application. The paralytic effect is maintained for 3-4 months, while in the autonomic
synapses the effect lasts for approximately 1 year (4). As already mentioned, excellent
safety profile is one of the key features of BoNT/A use. The adverse effects can be related
to the paralysis outside the target muscle because of the local diffusion to adjacent
muscles. This depends on the volume and speed of injection, dose, and site of injection.
For example, dysphagia can be a side-effect in the treatment of cervical dystonia, while
ptosis or a “frozen” face could be seen in facial aesthetics (9,10). After local injections of
BoNT/A, the fraction which didn’t enter the neurons is probably diluted in the lymphatic
circulation and washed away from the injection sites, being unable to affect more distant
nerve endings because of too low concentration.
BoNT/A action on pain
An unexpected discovery that BoNT/A injection into glabellar lines reduces
migraine headaches, followed by positive results coming from randomized clinical trials,
led to regulatory approval of Botox for chronic (not episodic) migraine headaches in
2011 (7). BoNT/A is injected intramuscularly in 31-39 anatomical points distributed
across the corrugator, procerus, frontalis, temporalis, occipitalis, cervical paraspinal, and
trapezius muscle groups. BoNT/A is injected intramuscularly in 31-39 anatomical points
distributed across the corrugator, procerus, frontalis, temporalis, occipitalis, cervical
paraspinal, and trapezius muscle groups. BoNT/A reduces migraine frequency and
provides modest improvement of migraine symptoms. BoNT/A is investigated in other
pain conditions and shows promising results in neuropathic and some other chronic pain
disorders (11,12). In contrast to conventional and non-conventional analgesic drugs, long-
lasting analgesic effect after a single application (several months in humans, more than
15 days in animals) is main advantage of BoNT/A use. While the results are promising,
the quality level of evidence due to a low number of participants, lack of standardized
dosing and delivery protocols, is not persuasive enough to provide explicit guidelines for
pain physicians (12).
Basic research of the action of BoNT/A on pain during the last two decades led to
an important insight into the molecular mechanism of its antinociceptive action. BoNT/A
was investigated in different inflammatory and neuropathic pain models, as well as on
models of “mirror” pain of central origin (Table II).
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Table II Preclinical investigation of BoNT/A in some experimental inflammatory and
neuropathic pain models (according to review of Matak et al. (12)
Pain models Results
Inflammatory pain models:
- formalin-induced spontaneous pain
- carrageenan-induced hypersensitivity
and paw oedema
- capsaicin–induced hypersensitivity
and neurogenic inflammation
BoNT/A applied s.c. (3.5-7 U/kg) into the rat hind-paw pad
or intrathecally (1 U/kg) into the lumbar segment of the
spinal cord reduced:
- pain behaviours in the second inflammatory phase of the
formalin test;
- thermal and mechanical hypersensitivity but not the size of
local oedema in the carrageenan model;
- thermal and mechanical hypersensitivity but not the local
neurogenic inflammation in the capsaicin model
Neuropathic pain models:
- partial sciatic nerve transection
- streptozotocin-induced diabetic
polineuropathy
- paclitaxel-induced neuropathic
polineuropathy
- infraorbital nerve constriction injury-
induced trigeminal neuropathy
BoNT/A applied s.c. (3.5-7 U/kg) into the hind-paw pad or
intrathecally (1 U/kg) into the lumbar segment of the spinal
cord reduced:
- mechanical and thermal hyperalgesia induced by partial
sciatic nerve injury;
- hypersensitivity to mechanical and thermal stimuli induced
by streptozotocin and paclitaxel (pain reduction was
observed on both paws although BoNT/A was applied on
one side only)
BoNT/A applied s.c. (3.5 U/kg) into the vibrissal pad
reduced:
- mechanical hypersensitivity after partial infraorbital nerve
constriction injury (pain reduction was observed on both
sides of the head although BoNT/A was applied into the
whisker pad on one side only)
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In analogy with action in the neuromuscular junction, first, it was suggested that the
antinociceptive effect of BoNT/A is a consequence of inhibition of neurotransmitter
release from peripheral sensory nerve endings (13). However, a series of behavioral data
from experimental models demonstrated that the BoNT/A action on pain occurs primarily
in the central nervous system (CNS), where it inhibits neurotransmitter release from
central terminals of primary afferent neurons. This was confirmed by the
immunohistochemical detection of cleaved SNAP-25, using antibody specific for
BoNT/A-cleaved SNAP-25 (the product of BoNT/A proteolytic activity), within the
sensory regions of the brainstem or spinal segment associated with the peripherally
injected area (12,14). These experiments have also suggested that axonal transport of
BoNT/A from the peripheral site of application to the CNS is a prerequisite for its action
on pain (Figure 2).
Figure 2. Proposed mechanism of antinociceptive action of BoNT/A
Furthermore, it was demonstrated that BoNT/A modulates spinal opioid, GABA
and glutamate neurotransmitter systems, as well as microglial activation and signaling.
Attenuation of the microglia activation and neuroinflammation was proposed to play a
role in the overall antinociceptive action of BoNT/A (12,15,16).
In the sensory system, BoNT/A action may be limited to certain neuronal
populations mediating nociception, as already demonstrated for capsaicin-sensitive
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neurons (17). This could explain the observed selective BoNT/A's antinociceptive action
on some types of chronic pain and patient subpopulations. Long-lasting action after a
single application is most likely a consequence of BoNT/A cellular localization and
escape of intracellular degradation (12). It was demonstrated that BoNT/A light chain
does not distribute evenly within the cytosol, but is concentrated at the inner side of the
plasma membrane thus interacting with small GTP-ase proteins that polymerize into non-
polar filaments to form a part of cytoskeleton. Another possible explanation is that
BoNT/A escapes the ubiquitine-proteasome degradation pathway by recruiting
specialized enzymes that remove polyubiquitin chains (12).
Further experiments investigating BoNT/A effects on multiple sites on its way from
the periphery to the CNS are needed to explain in more depth its action on pain of different
etiologies and to improve its clinical use.
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Botulinski toksin tip A: osnovni farmakološki
profil i terapijska primena
Lidija Bach-Rojecky*, Višnja Drinovac Vlah
Univerzitet u Zagrebu - Fakultet za Farmaciju i Biohemiju, Katedra za Farmakologiju,
Domagojeva 2, 10 000 Zagreb, Hrvatska
*Autor za korespondenciju: Lidija Bach-Rojecky, e-mail: lbach@pharma.hr
Sažetak
Botulin toksin tipa A (BoNT/A) proizvodi gram-pozitivna anaerobna bakterija Clostridium
botulinum i jedan je od najpotentnijih toksina u prirodi, ali i vrlo korisno terapijsko sredstvo kod
različitih neuroloških i autonomnih poremećaja. Glavne farmakološke karakteristike BoNT/A su
neurospecifičnost, dugotrajno dejstvo i sigurnost. Te su karakteristike utemeljene na njegovom
posebnom molekularnom mehanizmu delovanja: nakon specifičnog vezanja za membranu
neurona, internalizuje se u citosol, gde specifično cepa jedan od proteina potrebnih za egzocitozu
neurotransmitera. Posledična reverzibilna neuroparaliza traje nekoliko meseci i objašnjava
dugotrajne kliničke učinke nakon jednokratne lokalne primene toksina. BoNT/A je odobren za
primenu u prevenciji hronične migrene, a brojna pretklinička i klinička ispitivanja pokazala su
njegov potencijal u ublažavanju drugih hroničnih bolnih stanja. Mnogobrojni podaci iz
eksperimentalnih modela bola pokazali su da BoNT/A smanjuje patološku preosetljivost na bol
nakon aksonskog transporta u centralni nervni sistem, gde interferira sa složenim procesima
centralne senzitizacije. Potrebna su dodatna istraživanja koja bi detaljnije opisala molekularni
mehanizam delovanja BoNT/A na bol, kao i posebne farmakokinetičke karakteristike.
Ključne reči: botulinski toksin tipa A, mehanizam delovanja, terapijska primena,
ispitivanje bola
