Mechanisms of Botulinum Toxin Type A Action on Pain

Abstract

Already a well-established treatment for different autonomic and movement disorders, the use of botulinum toxin type A (BoNT/A) in pain conditions is now continuously expanding. Currently, the only approved use of BoNT/A in relation to pain is the treatment of chronic migraines. However, controlled clinical studies show promising results in neuropathic and other chronic pain disorders. In comparison with other conventional and non-conventional analgesic drugs, the greatest advantages of BoNT/A use are its sustained effect after a single application and its safety. Its efficacy in certain therapy-resistant pain conditions is of special importance. Novel results in recent years has led to a better understanding of its actions, although further experimental and clinical research is warranted. Here, we summarize the effects contributing to these advantageous properties of BoNT/A in pain therapy, specific actions along the nociceptive pathway, consequences of its central activities, the molecular mechanisms of actions in neurons, and general pharmacokinetic parameters.

Full text

toxins
Review
Mechanisms of Botulinum Toxin Type A Action
on Pain
Ivica Matak 1, *, Kata Bölcskei 2,3, Lidija Bach-Rojecky 4and Zsuzsanna Helyes 2,3
1Department of Pharmacology, University of Zagreb School of Medicine, Šalata 11, 10000 Zagreb, Croatia
2Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Szigeti út 12,
7624 Pécs, Hungary
3János Szentágothai Research Center, Center for Neuroscience, University of Pécs, Ifjúságútja 20,
7624 Pécs, Hungary
4Department of Pharmacology, University of Zagreb Faculty of Pharmacy and Biochemistry, Domagojeva 2,
10000 Zagreb, Croatia
*Correspondence: ivica.matak@mef.hr; Tel.: +385-1-456-6843
Received: 14 July 2019; Accepted: 29 July 2019; Published: 5 August 2019


Abstract:
Already a well-established treatment for different autonomic and movement disorders,
the use of botulinum toxin type A (BoNT/A) in pain conditions is now continuously expanding.
Currently, the only approved use of BoNT/A in relation to pain is the treatment of chronic migraines.
However, controlled clinical studies show promising results in neuropathic and other chronic pain
disorders. In comparison with other conventional and non-conventional analgesic drugs, the greatest
advantages of BoNT/A use are its sustained effect after a single application and its safety. Its efficacy
in certain therapy-resistant pain conditions is of special importance. Novel results in recent years has
led to a better understanding of its actions, although further experimental and clinical research is
warranted. Here, we summarize the effects contributing to these advantageous properties of BoNT/A
in pain therapy, specific actions along the nociceptive pathway, consequences of its central activities,
the molecular mechanisms of actions in neurons, and general pharmacokinetic parameters.
Keywords:
botulinum toxin type A; pain therapy; migraine; neuropathic pain; mechanism of action
Key Contribution: The analgesic effects of BoNT/A; specific actions along the nociceptive pathway;
consequences of its central activities; the molecular mechanisms of actions in neurons; as well as
general pharmacokinetic parameters are summarized.
1. Introduction
Botulinum toxin type A (BoNT/A) is among the most potent biological toxins in nature, and,
together with serotypes B, E, and F, a cause of natural botulism in humans, which is characterized by
flaccid paralysis of skeletal muscles and dysautonomia [1]. Based on early 19th century observations
that a yet unknown toxin from contaminated food induced the symptoms of botulism, the idea
emerged that a low dose of the agent could be used for overactive nerve disorder treatment. In the
20th century, different BoNT serotypes have been characterized and isolated, and Burgen et al.
discovered that BoNT/A inhibits acetylcholine release from neuromuscular junctions (NMJs) in skeletal
muscles [
2
]. These discoveries eventually lead to the development of its therapeutic use in the 1970s,
when minuscule quantities of the toxin were first employed to correct the eye misalignment in
strabism [
3
]. Today, botulinum toxin is the most commonly used therapeutic protein for the treatment
of autonomic disorders, spasticity and hyperkinetic movement disorders, as well as in cosmesis for
treating wrinkles [
4
]. BoNT/A is approved in upper limb spasticity, blepharospasm, hemifacial spasm,
cervical dystonia, primary hyperhidrosis, and neurogenic detrusor overactivity [4,5].
Toxins 2019,11, 459; doi:10.3390/toxins11080459 www.mdpi.com/journal/toxins

Toxins 2019,11, 459 2 of 24
BoNT/A effects on pain were reported in cervical dystonia in 1986 [
6
]. Initially, the analgesic effect
in neuromuscular disorders and musculoskeletal pain was attributed to the muscle relaxant effect,
until the antihyperalgesic effect in non-muscular pain models was unequivocally demonstrated in
human patients and animal models [
7
]. A serendipitous discovery that BoNT/A injection into glabellar
lines resulted in migraine resolution led to extensive investigation in headache, which demonstrated
that BoNT/A treatment was beneficial in chronic, but not in episodic migraine [
8
,
9
]. Efficacy in chronic
migraine was shown in two large industry-sponsored randomized controlled clinical trials (RCT),
after which onabotulinumtoxin A (Botox
®
) eventually gained regulatory approval in 2011 [
10
]. So far,
chronic migraine remains the only approved pain indication, even though several systematic reviews of
clinical trials provide evidence for BoNT/A efficacy in different pain conditions [
11
]. Nevertheless, we
have to add that, while the results are promising, the quality level of evidence is not yet high enough
to provide explicit guidelines for pain physicians. Shortcomings of the available clinical research data
include low number of participants, and thelack of standardized dosing and delivery protocols, as well.
In the present review, we summarize the available data on the mechanisms of action of BoNT/A
on pain, considering both peripheral and central mechanisms along the nociceptive pathways. We also
include an overview on the clinical evidence for chronic migraine and neuropathic pain syndromes.
There is ample preclinical evidence that peripheral and central sensitization is effectively and safely
alleviated by BoNT/A, but more clinical research is needed to determine the role of BoNT/A in chronic
pain management.
2. Basic Pharmacology of BoNT/A: Mechanisms of Outstandingly High BoNT/A Potency and
Long-Lasting Duration of Action
To describe how the effect of BoNT/A persists for months after its single use, it is important
to understand the structure and function of the different toxin protein subunits, which mediate its
high affinity recognition of neuronal targets and its intracellular enzymatic activity targeting the
synaptosomal-associated protein 25 (SNAP-25), a part of heterotrimeric soluble N-ethylmaleimide
sensitive factor attachment protein receptor (SNARE) complex (Sections 2.1–2.3). Apparently, the most
important factor regulating the longevity of toxin action is the ability of BoNT/A protease to avoid
cellular degradation mechanisms and survive in the cell cytoplasm for a long period (Section 2.4).
In Table 1we summed up the general factors affecting the BoNT/A potency and selectivity in relation
to its pharmacological properties, as well as possible improvement of its use.
Table 1.
General and pain-specific factors influencing the properties and peculiarities of botulinum
toxin type A (BoNT/A) action.
General Factors not Specific to Pain
Property of
BoNT/A Molecule
or Peculiarity of
Action
Mechanisms of Action Contribution to Desirable
Pharmacological Properties
Attempted or Potential
Improvement References
Low local diffusion
after application
rapid and high affinity
binding to neuronal
membrane at the
injected site
safety, onset of the effect lower injection volume,
intradermal injections [12–14]
Absorption
through epithelial
barriers
crossing epithelial barriers
by transcytosis
application by different routes
(e.g., transmucosal)
novel therapeutic systems
with incorporated BoNT/A
which might improve
toxin absorption and
extend the contact time
with the epithelial
tissue/mucosa
[15]
Specificity for
hyperactive
neurons
Expression of membrane
acceptors such as
glycosylated SV2C; higher
rate of SVs exo/endocytosis
favors toxin uptake
safety, selectivity for hyperactive
nerve terminals
recombinant chimeras
with different neuronal
specificities
[16]

Toxins 2019,11, 459 3 of 24
Table 1. Cont.
General Factors not Specific to Pain
Property of
BoNT/A Molecule
or Peculiarity of
Action
Mechanisms of Action Contribution to Desirable
Pharmacological Properties
Attempted or Potential
Improvement References
Protease specific
targeting of
SNARE proteins
synaptic localization,
disturbance of SNARE
supercomplex
potency, safety
- specific point mutations
for higher affinity to
SNAP-25
[17]
- recombinant molecules
with shorter action or
different affinity for
SNARE proteins
Protease longevity
inside neurons
cellular localization,
avoidance of proteasomal
degradation
long duration of effects
specific chimeras that
change the affinity for
intraneuronal degradation
system
[18]
Reversibility of the
neuroparalysis
recovery of neuronal
exocytosis is dependent on
nerve terminal type (gain
of function)
long duration of effect (from 3
months to more than a year)
interference with the nerve
function recovery
processes
[19]
Repeatability of
neuroparalysis
recovery of neuronal
exocytosis can be repeated
many times without loss of
neuron function
application schedule
repeated application for
prolonged period into the
same site
[20]
Factors Specifically Related to Pain
Property of
BoNT/A Molecule
or Peculiarity of
Action
Mechanisms of Action Contribution to Desirable
Pharmacological Properties
Attempted or Potential
Improvement References
Selectivity for
certain sensory
neuron populations
occurrence in
TRPV1-expressing neurons
selectivity for chronic or
prolonged pain recombinant chymeras
with different receptor
specificities
[21–23]
effect on glutamatergic
transmission
efficacy in chronic pain (possibly
LTP related)
effect on peptidergic
transmitters
efficacy in chronic pain and
migraine
Segmental activity
in the sensory
nucleus/spinal cord
dorsal horn
microtubule-dependent
neuronal axonal transport localization of toxin effect
neural block for segmental
treatment [24–26]
Interaction with
other pain
neurotransmitter
system
Interaction with
endogenous opioid system
synergism with opioid analgesic
and avoidance of tolerance
development; efficacy in
opioid-overused patients
combined use of lower
dose opioids and BoNT/A[27–29]
Restoration of sensitivity
to morphine
2.1. Structure of the BoNT/A Complex and Neurotoxin
From the lysed rod-shaped anaerobic bacterium Clostridium botulinum, BoNT/A is released as a large
900 kDa complex consisting of a 150 kDa neurotoxin and auxiliary proteins, which includes a non-toxic
non-hemagglutinin (NTNH) and three hemagglutinin proteins. The NTNH part of the progenitor
complex molecule contributes to toxin stability in the acidic environment and provides protection against
proteases in the gastrointestinal system, while haemaglutinins are involved in BoNT/A translocation
across the intestinal epithelial lining into the lymphatic system and the bloodstream [
30
]. Pharmaceutical
formulations may consist of the entire complex (onabotulinumtoxinA), some of its components (500 kDa
abobotulinumtoxinA), or the 150 kDa neurotoxin component only (incobotulinumtoxinA). It seems
that the non-toxic components rapidly dissociate from the neurotoxin at the neutral pH within the
injection site [
31
], and formulations containing the entire complex or only the neurotoxic component
exhibit the same potency [
32
]. Thus, the only component of the complex that contributes to its efficacy
upon injection seems to be the 150 kDa toxin [32].
The 150 kDa neurotoxin is a typical bacterial AB-structured toxin, consisting of heavy chain with
membrane acceptor–binding and translocation domains, and a smaller 50 kDa light chain, a catalytic
domain that mediates the intracytosolic proteolytic activity of the neurotoxin [33].

Toxins 2019,11, 459 4 of 24
2.2. Pharmacokinetics of Injected BoNT/A
As mentioned, at the injection site under neutral pH, non-toxic components rapidly dissociate from
the neurotoxin part of the molecule. The 150 kDa neurotoxin molecule, based on the injection technique
and volume, spreads away from the injection site, which may induce side effects if non-targeted
structures are reached. Examples include dysphagia in the treatment of cervical dystonia and paralysis
of unwanted muscles after cosmetic use leading to ptosis. In pain therapy, BoNT/A is usually employed
as a subcutaneous injection into multiple sites within the painful area, and intramuscular application
into the multiple head and neck sites is approved for migraine treatment. Intradermal BoNT/A injection
can also be used with the lower possibility for unwanted diffusion into nearby muscles compared
to subcutaneous or intramuscular injection [
34
,
35
]. Perineural or nerve block treatments have also
been suggested as effective modes of administration [
25
,
26
]. However, up to now, there have been no
controlled studies that compare the analgesic potency or efficacy following different injection modes.
It is known that, after oral or inhalational exposure, the toxin can reach the systemic circulation by
transcytotic transport across epithelial cells and further distributed to sensitive microcompartments,
such as the proximity of cholinergic nerves endings. However, the systemic pharmacokinetics of
BoNT/A used for therapeutic purposes (at doses not exceeding a few nanograms) has not been
resolved, mostly because of too low toxin concentrations and the limitations of detection in circulating
fluids. After local injections in tissues like the muscle, dermis, or subcutis, BoNT/A binds to the
neuronal membrane and enters the neurons, while the unbound fraction is probably diluted in the
lymphatic circulation and washed away from the injection sites, thus being unable to affect more
distant neuronal endings because of too low concentration [
33
]. The time-course of BoNT/A entrance
into peripheral neurons
in vivo
has been characterized and described only for larger, non-therapeutic
doses, showing that BoNT/A might poison the peripheral motor terminals within minutes after its
systemic injection [14]. Following radioiodinated BoNT/A injection into the muscle, the radioactivity
was reduced to control levels within 12 h, suggesting that the toxin must enter the peripheral terminals
within several hours to exert its paralytic activity before being diluted or degraded [
12
]. From the
therapeutic point of view, it is very important to elucidate the distribution (a transport via axons and
between cells) and degradation (pathways, enzymes, speed, and half-life) of both heavy and light
chains. These could elucidate differences in duration of the BoNT/A action observed between species
(human vs. mice/rat) and cell types (neuromuscular junction vs. autonomic cholinergic synapse vs.
sensory nerves).
2.3. Specificity of BoNT/A Effect: Acceptor-Mediated Entrance into the Neuronal Cytosol
BoNT/A exerts high tropism for neurons based on its specific binding sites on neuronal terminals
within the injected area. This specificity is due to a high affinity interaction of heavy chain and
its neuronal binding sites. Heavy chain C-terminus (H
C
) binds to double acceptors consisting of
gangliosides and synaptic vesicle 2 (SV2A-C) protein isoforms expressed at the extracellular side of
the neuronal membrane. The ganglioside with higher affinity for BoNT/A, such as GD1a and GT1b,
provide the surface for initial binding of BoNT/A (via heavy chain C-terminus) to neurons. Binding to
SV2 protein enables the toxin’s further endocytotic entrance into endosome-like compartments, such
as acidic synaptic vesicles (see detailed review by Pirazzini et al. [
33
]). The N-terminus of the heavy
chain (H
N
) may also be involved into the specific neuronal binding via interaction with phosphatidyl
inositol phosphates at the presynaptic plasma membrane [36].
It appears that the larger fraction of BoNT/A holotoxin undergoes sorting into acidic vesicles
leading to prompt translocation into the cytosol, while the smaller fraction that enters non-acidic
vesicular compartments may be sorted into the microtubule-dependent retrograde axonal transport
pathways [
37
,
38
]. Currently, it is not known if the transport to non-acidic vesicles might be mediated
by protein acceptors other than SV2. So far, BoNT/A has been demonstrated to enter the cell by binding
to fibroblast growth factor receptor 3 [
39
], and possible interaction has been reported with the transient
receptor potential vanilloid 1 (TRPV1) receptor activated by capsaicin and other vanilloid compounds,

Toxins 2019,11, 459 5 of 24
noxious heat, and lipid mediators [
40
]. The toxin light chain is released into the cytosol by energy- and
pH-dependent pore-forming process involving thioredoxin thioreductase [
33
]. During the process, the
light chain is separated from the heavy chain by reduction of the disulfide bridge and translocated
through the transmembrane pore formed by the HN.
2.4. Longevity of Light Chain-Mediated Enzymatic Activity
The 50 kDa domain light chain is the catalytic domain that cleaves the SNAP-25 molecules.
Naturally, any foreign protein in the cellular cytosol is the subject for proteasome-mediated degradation.
BoNT serotype E (BoNT/E) is rapidly degraded in the cytosol with its light chain intracellular half-life
being 1–2 days, leading to resolution of BoNT/E-mediated paralysis once the new SNAP-25 is
synthesized. Unlike BoNT/E, which distributes evenly within the cytosol by diffusion, BoNT/A
protease is concentrated at the inner side of the plasma membrane. It appears that double-leucine
motif is the determinant of such BoNT/A localization. The binding of BoNT/A near the synaptic
membrane involves interaction with septins, small GTP-ase proteins that polymerize into non-polar
filaments to form a part of cytoskeleton [
41
]. Another possible explanation is that BoNT/A escapes
the ubiquitine-proteasome degradation pathway by recruiting specialized enzymes that remove
polyubiquitin chains [
42
]. The enzymatic activity of BoNT/A persists for up to one year in neuronal
cultures, and up to five months in central neurons
in vivo
[
43
]. In comparison, the functional recovery of
the NMJ and the duration of the antinociceptive activity of BoNT/A are shorter in humans (3–4 months),
or even shorter in animals (around two weeks to one month). The duration of the neuromuscular
paralysis depends on the dose, suggesting that higher dose leads to uptake of higher number of
intracellularly active proteases in the cell [
44
]. In the sensory system, the effect of BoNT/A applied
subcutaneously in the rat hind paw lasted for 15 to 25 days [45,46].
It was hypothesized that one of the major contributing factors to the long-term BoNT/A effect is
the persistence of inactive SNARE heterotrimer in the presynaptic cleft [
47
]. However, after blockage of
proteolytic activity by intracellularly delivered electroporated antibodies, the synaptic neurotransmitter
release recovers after 4–5 days [
48
]. This time period is roughly similar to the period required for the
turnover of synaptic SNAP-25, suggesting that turnover of SNAP-25(1–197) has a similar rate [
49
]. Thus,
although SNAP-25(1–197) persistence within inactive SNARE heterotrimers might be contributing to
the high potency of BoNT/A, the long-term efficacy is most likely affected by the persistence of the
BoNT/A protease in the cell.
2.5. Inhibition of Neurotransmitter Release and the Effect on SNARE Supercomplex
Upon translocation into the cytosol, the light chain Zn
2+
-dependent metalloprotease enzymatically
cleavs one of the conserved cleavage sites in the SNAP-25 polypeptide (SNARE motifs). BoNT/A
and BoNT/E cleave the SNAP-25 at different SNARE motifs, forming truncated products of different
lengths. The BoNT/A-truncated product termed SNAP-25 (1–197) is lacking only nine amino-acids at
the C-terminus, while BoNT/E-cleaved product (SNAP-25(1–180) lacks 26 residues. Unlike BoNT/B and
BoNT/E, whose efficacy is dependent on the disrupted formation of the SNARE heterotrimer consisting
of SNAP-25, syntaxin, and VAMP-2/synaptobrevin, it appears that BoNT/A-mediated SNAP-25
cleavage does not affect the formation of the heterotrimer. Possibly due to preserved interaction with
other SNAREs, SNAP-25(1–197) is not readily cleared away from the presynaptic terminal unlike
BoNT/E-truncated product SNAP-25(1–180) [
50
]. In turn, SNARE-heterotrimer complex containing
SNAP-25(1–197) competes with normal SNARE complexes at the vesicular release site, which is
supported by finding that recombinant SNAP-25(1–197) inside the cell leads to neurotransmitter release
blockade by producing a membrane-bound product [
51
]. This explains the disproportionate percent
inhibition of synaptic neurotransmitter release evoked by BoNT/A in comparison to percentage of
cleaved SNAP-25 molecules [
49
].
In vitro
investigations suggested that only a small fraction (2–20%) of
SNAP-25 molecules leads to complete synaptic blockade [
52
,
53
]. This implies that BoNT/A, by cleaving
only a small subset of SNAP-25 molecules, blocks the transmitter release completely. Possibly, only

Toxins 2019,11, 459 6 of 24
1–2 molecules of BoNT/A light-chain per synapse may be enough for the blockage of entire synaptic
release machinery at individual synapses. Another factor in BoNT/A exquisite potency is the fact that
the vesicle release site involves a formation of radial super-complex of SNARE heterotrimers, acting in
concert to release one synaptic vesicle at a time. It appears that cleavage of only one or two molecules
of SNAP-25 within the SNARE supercomplex disrupts the entire supercomplex function [
54
]. Thus, it
is likely that two major factors affecting its potency are (1) specific binding of neuronal terminal and (2)
disruption of small but vital population of synaptic SNAP-25 at the site of transmitter release.
2.6. Selectivity for Excitatory Synapses and Ca2+Dynamics
Blockage of neurotransmitter release leads to build-up of synaptic vesicles near the presynaptic
membrane. Apparently, BoNT/A is highly potent to block acetylcholine release and less potent to inhibit
most other neurotransmitters such as glutamate, noradrenaline, serotonin, substance P, calcitonin
gene-related peptide (CGRP), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD),
etc. [
33
,
55
,
56
], which might depend on the level of expression of high-affinity protein acceptors in the
neuronal membrane.
SNARE-mediated vesicular release machinery requires a Ca
2+
-mediated signal to undergo
conformational changes leading to vesicular membrane fusion with presynaptic plasma membrane.
This is provided by interaction of vesicle-associated calcium sensor protein synaptotagmin with the
C-terminal of SNAP-25 [
57
]. The main effect of BoNT/A-mediated SNAP-25 cleavage is a reduced affinity
of intracellular Ca
2+
sensor synaptotagmin to SNAP-25. Under normal, physiological concentration of
Ca
2+
, the lack of 9 amino-acids at the C terminus of SNAP-25(1–197) impairs this interaction. However,
the interaction is restored under high Ca
2+
concentration, and SNARE complex regains its normal
neurosecretory function [
58
]. Thus, any treatment aimed at increasing the Ca
2+
concentration restores
the neurosecretory function of BoNT/A-poisoned synapse, which may be responsible for observed lack
of BoNT/A effect on capsaicin-evoked release of peptides in few in vitro studies [59,60] (see ref. [61]).
Regarding the
in vitro
release of inhibitory neurotransmitters, in the mouse embryonic spinal
cord, BoNT/A appears to block the evoked release of glycine at similar potency compared to ACh.
However, it appears that BoNT/A does not block the release of GABA in adult neurons. It has been
posited that inhibitory neurons do not express SNAP-25, and that the neurotransmitter release in
GABA-ergic neurons might be mediated by some other SNAP-25 isoform (e.g., SNAP-23) [
62
]. However,
GABA-ergic neurotransmitter release is also invariably modulated by SNAP-25, since gene deletion of
SNAP-25 expression abolishes the neurotransmitter release in GABA-ergic neurons. It appears that the
effect of SNAP-25 cleavage is readily overcome by transient synaptic increase of intracellular calcium
in adult inhibitory neurons. Interestingly, the level of SNAP-25 expression appears to be contributing
to the calcium levels, since it interacts with ion channels as a negative regulator on voltage-gated
calcium channels [
63
]. Artificial expression of high levels of SNAP-25, or application of Ca
2+
chelators,
confers GABA-ergic neurons more sensitive to BoNT/A. Thus, the level of SNAP-25 contributes to the
low sensitivity of inhibitory neurons and, conversely, higher sensitivity of glutamatergic synapses to
BoNT/A due to additional function of SNAP-25, which alters the calcium dynamics via its interaction
with Ca2+channels [64].
2.7. Interaction with Ion Channels and Pain-Sensing Receptor Translocation
By inducing cleavage of SNAP-25, BoNT/A may interfere with protein translocation from
endosomal compartment to the cell plasma membrane. This has been proven for the TRPV1 capsaicin
receptor, which is an important non-selective cation channel in pain transmission [
65
,
66
]. BoNT/A
might interfere with the function of Na
+
channels as well [
67
], possibly also due to this mechanism.
Based on investigation of mechanical sensibility of dural afferents, it was proposed that BoNT/A
may decrease the activity of mechanosensitive receptors and the transient receptor potential ankyrin
1 (TRPA1) channels [
68
,
69
] (their molecular identity not being characterized in mentioned studies).
These mechanisms have been proposed to contribute to the BoNT/A antinociceptive activity.

Toxins 2019,11, 459 7 of 24
3. BoNT/A Effects on Peripheral Sensory Nerves
3.1. Prevention of Nociceptive Neurotransmitter Release in Peripheral Terminals
While it was initially believed that BoNT/A antinociceptive effects are mediated by its actions on
the muscles, the findings that a broad range of pain conditions not related to muscular contraction were
also relieved by BoNT/A have soon suggested its possible effects on the sensory neurons. In analogy
with its known action on the neuromuscular junction, it was proposed that BoNT/A prevents the
sensory neurotransmitter release from peripheral sensory nerve endings. There is a plethora of studies
that demonstrated the blockade of nociceptive neurotransmitter release by BoNT/A
in vitro
from
peripheral sensory nerves. In primary sensory neuronal cultures, BoNT/A blocks the KCl-evoked
release of CGRP and substance P [
61
,
70
], suggesting that sensory neuropeptide release is dependent on
the SNARE complex. This has been confirmed in ex vivo urinary bladder preparations [
71
,
72
]. Studies
involving formalin-induced stimulation of rat hind-paw and temporomandibular joint reported a
reduced elevation of tissue content of glutamate and substance P by peripherally injected BoNT/A [
7
,
73
].
In human skin, intradermally injected BoNT/A reduces the capsaicin- and heat-evoked glutamate
release measured by microdialysis [
74
]. Although BoNT/A prevents peripheral nociceptive transmitter
release, preclinical data provided no evidence that such peripheral toxin action is causally involved in
its antinociceptive effect. Moreover, it was shown that BoNT/A antinociceptive action is not causally
related to toxin’s anti-inflammmatory effects, which are presumably mediated by prevention of
peripheral neurotransmitter release, either (Section 3.2). In addition, antinociceptive effect of BoNT/A
was demonstrated in centrally-mediated pain models (Section 5).
3.2. Anti-Inflammatory Effects of BoNT/A
In contrast to consistent evidence for BoNT/A inhibitory action on pain of different etiologies,
BoNT/A’s effect on inflammation is still inconclusive, mostly because of contradictory animal
experimental results. Cui et al. [
7
] were the first to show that intraplantar BoNT/A injection reduced
formalin-induced edema and accompanied peripheral glutamate release, thus, suggesting inhibition of
peripheral sensitization as the primary mechanism of BoNT/A action on pain and inflammation. In
contrast, in acute inflammatory pain models evoked by intraplantar injection of carrageenan or capsaicin,
BoNT/A pretreatment reduced pain hypersensitivity but failed to affect either carrageenan-induced
paw edema or capsaicin-induced plasma protein extravasation both at macroscopic and histological
levels [
75
]. The observed dissociation between the effects on pain and local inflammation questioned
the well-established concept about the common peripheral mechanism of BoNT/A action. In models
of cyclophosphamide-induced cystitis and capsaicin-induced prostatitis, local administration of
BoNT/A decreased bladder hypersensitivity and neurogenic inflammation (measured as decreased
concentrations of CGRP and SP) as well as inflammatory cell accumulation and cyclooxygenase-2
expression in the prostate and spinal cord [
76
,
77
]. Furthermore, the anti-inflammatory action of BoNT/A
was tested in models of acute or chronic arthritis. Reduction of long-lasting complete Freund’s adjuvant
(CFA) - induced joint inflammation and destruction shown by decreased inflammatory cell infiltration
around the articular cartilage and synovial membrane were observed for two weeks after intraarticular
BoNT-A application [
78
]. Additionally, intraarticular BoNT/A injection decreased CFA-induced
expression of the proinflammatory cytokines IL-1
β
or TNF-
α
in the synovial tissue, accompanied by
alleviation of cartilage degeneration and inflammatory cell infiltration [
79
]. Intraarticular BoNT-A
reduced the persistent inflammatory hypersensitivity induced by systemic CFA and intraarticular
methylated BSA in the temporomandibular joint as well, and significantly diminished the peripheral
release of the SP, CGRP, and IL-1β[73].
Human data about the peripheral anti-inflamatory effects are also inconsistent. Bittencourt da
Silva et al. found a reduction of glutamate release in dermal microdialysates by BoNT/A injections in
healthy volunteers after capsaicin and thermal provocation [
74
]. In contrast, Attal et al. [
45
] in skin
punch biopsy specimens of patients with peripheral neuropathic pain found no difference in SP and

Toxins 2019,11, 459 8 of 24
CGRP content between the BoNT/A and the saline-treated control group (however, in the mentioned
study, CGRP and SP content was not compared to normal healthy controls). While the first study
proposed the involvement of transmitter release inhibition in the analgesic action of BoNT/A, the
second study questioned the role of peripheral neuropeptides in the toxin’s analgesic effect, at least in
peripheral neuropathic pain.
Important insight into the mechanism of antinociceptive action of BoNT/A came from a set of
experiments on neuropathic pain models, whose main findings are shown in Table 2.
Table 2. Key findings from neuropathic pain models.
Model BoNT (Type; Dose,
Application) Findings/Comments Ref.
partial sciatic nerve
injury in rats
A; 7 U/kg; i.pl.; injected
after established
hypersensitivity (day 14)
Long-term reduction of mechanical and thermal hypersensitivity
(from day 5 after injection). First study on experimental peripheral
neuropathic pain.
[45]
ligation of L5/L6 spinal
nerve in rats
A; 10, 20, 30 or 40 U/kg
i.pl. after established
hypersenitivity
Reduction of mechanical allodynia (after 1 day) and cold allodynia
(three days after injection; both effects lasted for 15 days after
injection). The effect was dose-dependent. However, large systemic
doses were used.
[80]
chronic constriction
injury of the sciatic nerve
in mice
A; 15 pg/mouse; i.pl. pre-
and post-injury
Reduced mechanical allodynia (from day 1 after injection; lasting at
least three weeks). BoNT/A reduced pain symptoms only if injected
after neuropathy onset, but not as a pretreatment.
[81]
paclitaxel-induced
peripheral
polyneuropathy in rats
abobotulinumtoxinA
(AboA); 20-30 U/kg; i.pl;
post-treatment
Antihyperalgesic effect at both ipsilateral and contralateral paws
(three and six days after injection). [82]
streptozotocin diabetic
neuropahy in rats
A; 3, 5 and 7 U/kg (i.pl);
1 U/kg (i.t.);
post-treatment
Unilateral toxin application reduced mechanical and thermal
hypersensitivity bilaterally (from fifth to 15th day after BoNT/A).
Intrathecal BoNT-A was effective after 24h. Different onset and lower
analgesic dose after intrathecal injection suggested central action of
BoNT/A.
[83]
chronic constriction
injury to the sciatic nerve
in mice and in rats
A;1.875, 3.75, 7.5 and
15 pg/paw for mice; 18.
75 or 75 pg/paw or i.t. for
rats; post-treatment on
day 5
Single i.pl. or i.t. injection significantly reduced the mechanical
allodynia in mice and rats and thermal hyperalgesia in rats (from 24 h
after toxin injection) and lasted for several weeks). Acceleration of
regenerative processes in the injured nerve was also observed.
[84]
chronic constriction
injury of the sciatic nerve
in rats
A; 75 pg/paw; i.pl.;
3 days before and 5 days
after CCI
Reduced neuropathic pain-related behavior and attenuated
upregulation of NOS1, prodynorphin, pronociceptin mRNA in the
DRG and microglia activation in both the spinal cord and DRG.
[85]
L5 ventral root
transection (VRT) in rats
A; 7 U/kg; i.pl.;
post-injury 4, 8 or
16 days
Reduced mechanical allodynia bilaterally and inhibited P2X (3)
over-expression in DRG nociceptive neurons unulaterally to L5 VRT. [46]
Infraorbital nerve
constriction (IoNC)
in rats
A; 3.5 U/kg into vibrissal
pad; post-injury on
day 14
Unilateral toxin injection reduced the IoNC-induced dural
extravasation and allodynia bilaterally (from day 2 and lasting 17
days after BoNT/A, prior to neuropathy resolution). Intraganglionic
block of axonal transport by colchicine abolished the effects of
BoNT/A. Bilateral effects of BoNT/A and dependence on retrograde
axonal transport suggest a central site of its action.
[86]
transection of the L5
ventral root in rats
A; 10 or 20 U/kg, i.pl.
post-injury at day 3
Bilaterally decreased mechanical hyperalgesia, (from day 5, lasting at
least 20 days post-BoNT/A). BoNT/A lowered the VRT-induced
increased percentage of TRPV1 (+) neurons in the ipsilateral DRG.
[87]
chronic constriction
injury in mice
A; 15 pg/paw i.pl.;
post-injury at day 4
Counteracted allodynia and reduced astrocyte activation. It increased
the analgesic effect of morphine and countered morphine-induced
tolerance. In neurons BoNT/A restored the expression of MORs
reduced by repeated morphine administration.
[88]
partial sciatic nerve
transection in rats
A; 7 U/kg, i.pl.
post-injury at day 14
Decreased mechanical and cold allodynia. Opioid antagonist
naltrexone applied five days after the toxin reversed its
antinociceptive effect. Central antinociceptive action of BoNT/A
might be associated with the activity of endogenous opioid system
via µ-opioid receptor.
[27]
partial sciatic nerve
transection in rats
A; 7 U/kg, i.pl.
post-injury at day 14
Reduced mechanical allodynia. GABA-A antagonist bicuculine
abolished the antinociceptive effect in toxin-treated animals, thus
indicating involvement of central GABAergic system.
[89]
chronic constriction
injury of the infraorbital
nerve in rats
A; 3 or 10 U/kg; s.c. into
the whisker pad;
post-injuryt at day 14
The toxin exerted antinociceptive effect and significantly lowered the
expression of TRPA1, TRPV1, and TRPV2 in trigeminal nucleus
caudalis (Vc); these effects were blocked by colchicine.
[90]

Toxins 2019,11, 459 9 of 24
Table 2. Cont.
Model BoNT (Type; Dose,
Application) Findings/Comments Ref.
surgical constriction of
the infraorbital nerve
in rats
A; 15 U/kg; post-surgery
at day 6; injected into the
area of nerve ligation
Reduced thermal nociceptive response (TNR) beginning 6 h and
lasting 72 h after treatment in senzitized animals. BoNT/A in sham
group increased TNR thus suggesting a pronociceptive effect in
non-sensitized animals.
[91]
malpositioned dental
implants to induce injury
to the inferior alveolar
nerve in rats
A;1or3U/kg s.c. into
the facial region; 3 days
post-injury
Attenuated mechanical allodynia. Double treatments with 1 U/kg of
BoNT-A produced prolonged, more antiallodynic effects as compared
with single treatments. BoNT-A significantly inhibited the
upregulation of Nav1.7 expression in the trigeminal ganglion in the
nerve-injured animals.
[92]
chronic constriction
injury of the sciatic nerve
in rats
A; 300 pg/paw; i.pl.
post-surgery at day 5
Attenuated pain-related behavior and microglial activation. It
restored the neuroimmune balance by decreasing the levels of
pronociceptive factors (IL-1βand IL-18) and increasing the levels of
antinociceptive factors (IL-10 and IL-1RA) in the spinal cord and DRG.
[93]
streptozotocin-induced
diabetic polyneuropathy;
chronic constriction
injury in rats
aboA; 15 or 20 U/kg; s.c.;
post-injection and
post-injury at day 14
Unilateral aboA reduced bilateral mechanical hyperalgesia in diabetic
polyneuropathy model, while had no effect on unilateral CCI-induced
hyperalgesia if applied contralaterally to the injury.
[94]
rat spared nerve injury
(SNI) model
LC/E-BoNT/A chimera;
15–75 U/kg, i.pl.
post-surgery at day 4
Alleviated for ∼two weeks mechanical and cold hyper-sensitivities.
When injected five weeks after injury, LC/E-BoNT/A still reversed
fully-established mechanical and cold hyper-sensitivity.
[18]
partial sciatic nerve
ligation in mice (SP and
NK1R knockout mice)
A; 0.2 and 0.4 U/paw, i.pl.
post-surgery at day 7
Reduced hyperalgesia in wild type animals, but not in gene-deleted
groups, suggesting the necessary involvement of SP-ergic system in
the antinociceptive activity of BoNT/A.
[95]
3.3. Involvement of BoNT/A Systemic Effect in the Measurement of Nociceptive Responses
In the majority of animal studies described above, the antinociceptive effect of the toxin has been
studied by measuring motor responses, such as hind-paw withdrawal following non-painful or painful
mechanical or thermal stimuli. Generally, when assessing the analgesic efficacy of substances affecting
motor performance, the effect on the motor performance of the drug itself might confound the results
of nociceptive pain measurement. Due to possible BoNT/A diffusion from the site of toxin injection
into the bloodstream, especially at higher doses, some systemic effects might be expected to result in
decreased overall motor performance, and thus, possibly affecting the pain-evoked motor responses.
Interestingly, within low dose BoNT/A range in rats (3.5 U/kg–15 U/kg), it has been observed that the
pain-evoked motor response usually peaks with the lowest effective dose [
7
,
45
,
75
]. However, some
animal studies reported dose-response effect by taking into account high doses which most likely
affected the motor performance. In rats, Cui et al. [
7
] found reduction of formalin-evoked phase II
spontaneous response at 30 U/kg compared to 3.5–15 U/kg doses, and Park et al. [
80
] also reported
reduction of allodynia based on comparison of 10 U/kg, 20 U/kg, 30 U/kg, and 40 U/kg doses. Thus, in
studies involving BoNT/A, possible systemic effect of employed BoNT/A dose has to be ruled out first
by testing the motor performance after particular mode of toxin injection.
3.4. Regenerative Effects of BoNT/A in the Injured Nerve
An interesting set of observations from neuropathic pain model based on chronic constriction
injury (CCI) suggested that BoNT/A affects the functional recovery of injured peripheral nerves.
BoNT/A injected intraplantarly in neuropathic mice improved the sciatic index and weight bearing,
along with increased cell division cycle 2 (cdc2) protein expression and Schwann cell proliferation
and maturation [
84
,
85
]. Further study indicated that BoNT/A might be axonally transported within
the sciatic nerve trunk and enter the Schwann cells [
96
]. Interestingly, BoNT serotype B (BoNT/B),
which also counteracts the pain evoked by CCI, does not possess the nerve regenerative ability [
97
].
This suggests an additional role of cleaved SNAP-25 or some yet unknown effects of BoNT/A on gene
expression patterns within the injured nerve, which are not affected by BoNT/B.

Toxins 2019,11, 459 10 of 24
3.5. Effects of BoNT/A on the Sensory Ganglia
In the sensory ganglia of injured nerves, BoNT/A reduces the pain-evoked upregulated protein
expression of nociception-related ion channels such as TRPV1, purinoceptor P2X3, and reduces the
mRNA expression of pronociceptive peptides such as preprodynorphin [
46
,
85
,
87
]. Based on reduced
surface expression of the TRPV1 receptor protein, but not its mRNA, it was proposed that BoNT/A might
block the translocation of TRPV1 to the sensory neuronal surface in the ganglia. This was corroborated
by
in vitro
studies of primary sensory neuronal cultures [
65
,
66
]. In trigeminal ganglion primary
sensory neurons acutely isolated from animals with infraorbital nerve constriction (IoNC) and BoNT/A
or saline injection into the whisker pad, BoNT/A prevented the KCl-evoked neuroexocytosis [
98
].
Shimizu et al. [
66
] demonstrated that BoNT/A reduced the TRPV1 expression in sensory neurons
projecting from the dura mater. It was reported that BoNT/A might be axonally transported from
the periphery to dural CGRP-expressing primary afferents [
22
]. Since peripheral orofacial area and
dura are not innervated by the same sensory neurons, these findings indicate BoNT/A trans-synaptic
transport between sensory neurons that innervate different intracranial and extracranial targets [
22
,
99
].
This mechanism of BoNT/A traffic provides possible explanation for BoNT/A efficacy in migraine pain
associated with meningeal afferents. It was also hypothesized that BoNT/A, if transported into satellite
glial cells, might modulate the release of glutamate from glial cells interacting with sensory neurons
and alter the intraganglionic communication between the glia and sensory neurons [
100
]. However,
this possibility has not been examined in vivo.
BoNT/A injected directly into the rat trigeminal ganglion reduced the orofacial formalin-induced
pain [
101
], and infraorbital nerve constriction (IoNC)-induced trigeminal neuropathic pain [
86
].
Although the ganglia might be an important site of toxin action contributing to its antinociceptive
activity, it seems that BoNT/A action within the ganglia is not sufficient. Injection of colchicine into the
ganglion before the BoNT/A intraganglionic injection prevented the toxin effect on pain. In addition, if
the site of action was the ganglion, BoNT/A i.g. injection should have induced a fast antinociceptive
effect visible after 24 h. However, the analgesic action of such injection was delayed—it was evident
after two days. Thus, BoNT/A action on pain is dependent on axonal transport beyond the ganglion,
most likely into the CNS [86,101].
4. Actions of BoNT/A in the Central Nervous System
The antinociceptive effect of BoNT/A was investigated in several pain models wherein the unilateral
tissue injury induces a long-lasting bilateral pain hypersensitivity. In these models, it has been accepted
that pain development, its spread to contralateral side, and chronification is most probably mediated
by complex spinal and supraspinal mechanisms [
102
,
103
]. In the model of intramuscular (i.m.)
acidic saline induced “mirror pain,” BoNT/A injected into the ipsilateral hind paw pad significantly
reduces mechanical hypersensitivity on the injured but also on the uninjured contralateral side [
104
].
Bilateral effect of BoNT/A was repeatedly shown in other bilateral pain models, like in trigeminal
neuropathy induced by unilateral infraorbital nerve constriction injury (IoNC) [
86
], inflammatory
pain induced by complete Freund’s adjuvant (CFA) injection into temporomandibular joint [
22
], and
hyperalgesia after carrageenan injection into the calf muscle [
24
]. The toxin’s bilateral effect after
unilateral injection was demonstrated in poly-neuropathic states evoked by systemic paclitaxel [
82
]
or streptozotocin [
83
], as well as in the model of bilateral acute model of s.c. carrageenan-induced
inflammatory hyperalgesia [94].
When injected into the spinal canal, BoNT/A relieved pain faster and in lower doses (within
24 h, 1–2 U/kg) compared to local subcutaneous (s.c.) injection (3–5 days; 3.5–7 U/kg). However,
it was completely ineffective if applied supraspinally, e.g., to cisterna magna [
24
]. The effect on
bilateral pain depends on the axonal transport, since the axonal transport blocker colchicine prevented
the reduction of pain on both sides. These consistent behavioral observations strongly support the
BoNT/A central effect at the level of spinal cord segment associated with peripherally innervated area.
Immunohistochemical studies of BoNT/A-cleaved SNAP-25 demonstrated that BoNT/A is axonally

Toxins 2019,11, 459 11 of 24
transported into the sensory regions of brainstem or spinal segment associated with the peripherally
injected area. Following facial injection, BoNT/A-cleaved SNAP-25 was visible only in pain-associated
areas of the spinal trigeminal nucleus system (trigeminal nuclei oralis and caudalis [24,105].
Interestingly, BoNT/A application on the side contralateral to the injuries evoked by acidic saline
or carrageenan did not lead to bilateral action. In the model of acidic saline-induced chronic mechanical
hypersensitivity, contralateral BoNT/A reduced the pain only on the injection side [
104
]. On the other
hand, in carrageenan-induced mechanical hyperalgesia model, contralaterally injected BoNT/A failed
to affect pain on either side [
24
]. Based on these results, we may speculate that the bilateral effect of
BoNT/A depends on the type and mechanisms of the injury (intensity and substance used to provoke
tissue damage, time-course of bilateral pain development, etc). Observations of unilateral activity or
no activity after contralateral BoNT/A injection suggest that BoNT/A is not necessarily transported to
the contralateral side.
4.1. Effects in TRPV1 Receptor-Expressing Central Afferent Terminals
BoNT/A does not affect any other sensory sensation, apart from pain-related inflammatory and
mechanical stimulation. One of the possible explanations for this selectivity, in contrast to other sensory
modalities, is the selective BoNT/A entrance into particular sensory neuron population (Figure 1). It was
shown that the effects of BoNT/A on orofacial formalin-induced pain were prevented by the destruction
of TRPV1-expressing afferents evoked by high dose capsaicin injection into the trigeminal ganglion [
21
].
In addition, the SNAP-25 cleavage in the trigeminal nucleus caudalis was also abolished and prevented
by trigeminal denervation with capsaicin. This study suggested the occurrence of BoNT/A enzymatic
activity in central afferent terminals of capsaicin-sensitive TRPV1-expressing neurons (Figure 1).
The exact reason for this selectivity is not known, but the capsaicin-sensitive primary afferents might
be more prone to acceptor-mediated BoNT/A entrance at the periphery. It is also possible that sensory
neurons in neuropathic or other pain conditions are expressing higher levels of SV2A proteins, which
might facilitate the BoNT/A entry only into the sensitized sensory neurons [
106
]. In line with necessary
role of sensory neurons, patients with more allodynia or better preserved sensory function better
respond to the pain treatment with BoNT/A [
34
,
107
]. Moreover, the importance of capsaicin-sensitive
neurons is supported by the observation that neuropathic patients with lower thermal deficits responded
better to BoNT/A treatment [
107
]. In different homozygous knockout mice with gene deletions of
neurotransmitters and receptors related to capsaicin-sensitive neurons (TRPV1, NK1 receptor, and
substance P/neurokininA), BoNT/A failed to reduce the neuropathic and inflammatory pain [
21
,
95
].
These findings suggest important role of functional transmission within capsaicin-sensitive neurons
for exertion of BoNT/A antinociceptive activity. However, in bilateral carrageenan-induced chronic
mechanical hyperalgesia, the desensitization of sciatic nerve with inrasciatic high-dose capsaicin
treatment did not affect BoNT/A’s bilateral antinociceptive effect [
24
], nor did capsaicin itself affect
the contralateral carrageenan-evoked pain [
24
]. Thus, it may be speculated that, at least in sciatic
innervation area, BoNT/A might also require other types of neurons to achieve this bilateral effect.
4.2. Indirect Central Actions on the Endogenous Opioid and GABA Neurotransmission
Drinovac et al. [
27
,
89
], based on several lines of experiments on rats with pain in the sciatic region,
suggested that enhancement of the opioid and GABA neurotransmission mediated by their receptors
(
µ
-opioid and GABA-A) is involved in the central antinociceptive effect of BoNT/A. Although the
opioid receptor antagonist naltrexone, as well as the GABA-A antagonist bicuculline, reduced BoNT/A
effect on pain when applied intraperitoneally or intrathecally, the antagonistic effect was completely
absent after their intracisternal and intracerebroventricular injection, thus providing evidence for
segmental intraspinal action of BoNT/A on pain [
24
]. Furthermore, the analgesic action in the trigeminal
innervation region also involves interactions with the central, not peripheral, endogenous opioid
system, most likely at the level of trigeminal nucleus caudalis [
108
]. Interestingly, in the CCI-induced
neuropathic pain model of mice, BoNT/A co-administration increased the morphine-induced analgesic

Toxins 2019,11, 459 12 of 24
response and reduced the tolerance to repeated morphine application accompanied with enhanced
neuronal
µ
-opioid receptor expression [
88
,
109
]. It thus seems that modulatory spinal inhibitory
neurotransmitter system, which is known to attenuate sensory input to the dorsal horn, plays a
significant role in the central antinociceptive action of BoNT/A. However, the mechanism of this
interaction is still not clear. It possibly involves some yet unidentified neuronal circuits within the spinal
cord rather than direct action of BoNT/A on the inhibitory neurons. Opioid and GABA and transmissions
have a role in the attenuation of sensory input to the spinal dorsal horn. Similarly to BoNT/A, different
treatments suppress the morphine-induced pain tolerance, such as chronic N-methyl-D-aspartate
receptor (NMDAR), neurokinin 1 receptor (NK1), or TRPV1 antagonist treatments and desensitization
of capsaicin-sensitive neurons, etc. [
110
–
112
]. GABA receptor positive allosteric modulators, such as
ethanol and diazepam, also suppress morphine-induced tolerance, and their effects are reversible by
bicuculline [
113
]. It can be hypothesized that BoNT/A affects a common pathway inducing morphine
tolerance, e.g., synaptic transmission via glutamate from primary afferent terminals to the secondary
sensory neurons in the spinal dorsal horn. This might consequently induce a compensatory increase in
the morphine-suppressed endogenous opioidergic inhibitory neurotransmission. BoNT/A was shown
to be effective in headache patients resistant to analgesics and having medication overuse [
28
,
29
],
which is in line with its ability to restore the normal opioidergic transmission in the trigeminal nucleus
caudalis and spinal dorsal horn.
4.3. Effects on Astroglia and Microglia (Neuroinflammation)
Activated microglia and astrocytes have a well-known role in the progression and maintenance of
neuropathic pain [
85
]. Hence, a potential interference of BoNT/A with glial cells was investigated in
several neuropathic pain models in mice and rats (Table 2). Mika et al. [
85
] showed that intraplantar
BoNT/A injection reduced CCI-induced mechanical and thermal hypersensitivity and microglia (but
not astrocyte) activation in the ipsilateral lumbar spinal cords in rats. Attenuation of the microglia
activation and neuroinflammation was proposed to play a role in the overall antinociceptive action
of BoNT/A. Furthermore, in the same model in mice, it was demonstrated that intraplantar BoNT/A
injection reduced microglia activation and astrocyte activiation in both the dorsal and ventral horns
of the spinal cord [
88
]. In CCI-exposed rats Zychowska et al. [
93
] showed that BoNT/A diminished
microglia activation, the levels of the pro-inflammatory citokines IL-1
β
and IL-18, and enhanced the
concentrations of the inhibitory interleukins IL-1RA and IL-10 in the spinal cord and/or the DRG,
suggesting BoNT/A-mediated reinstatement of neuroimmune balance deteriorated by the nerve injury.
Those
in vivo
experiments drew attention to the involvement of glia in the antinociceptive effect
of BoNT/A, at least in neuropathic pain models, however, nothing was known about the nature
of this interaction. A recent
in vitro
study demonstrated that BoNT/A inhibited the expression of
pro-inflammatory IL-1
β
, IL-18, IL-6, and nitric oxide synthase 2 (NOS2) through the inhibition of
p38-, ERK1/2-, and NF-
κ
B-mediated intracellular signaling pathways on primary rat microglia, but not
astrocyte cell line after lipopolysaccharide stimulation. Additionally, it decreased the expression of
SNAP-23 (the main SNAP molecule in microglia) and increased TLR2 expression, which is suggested
to be its microglial molecular target [
114
]. These results are in line with previous observations on a
murine macrophage cell line, where BoNT/A induced changes of global gene expression through a
TLR2-dependent pathway [
115
] These results propose a direct interaction of BoNT/A with microglia
cells and reveal interference with some intracellular signaling processes as an explanation for
in vivo
findings. This topic needs further investigations to reveal the details of BoNT/A action on the
neuroinflammatory mechanisms.
4.4. Effects on the Ascending Pain Processing Pathway
As mentioned, the main site of the antinociceptive effect of BoNT/A is situated at the level of
segmental spinal dorsal horn and/or the brainstem sensory region, associated with the toxin-injected
area. Studies in the optic nervous system demonstrated the toxin’s sequential axonal transport and

Toxins 2019,11, 459 13 of 24
transcytosis over several synapses, similarly to tetanus toxin [
116
,
117
]. Thus, it is possible that BoNT/A,
following transcytosis in the medullary or spinal dorsal horn, might be transported into the ascending
sensory regions by a similar mechanism.
Up to now, this has been examined by Matak et al. [
21
]. After BoNT/A injection into the rat
whisker pad, cleaved SNAP-25 was found only in the spinal trigeminal nucleus caudalis and oralis,
but not in other sensory regions examined (thalamus, hypothalamus, sensory cortex, locus coeruleus,
periaqueductal gray, etc.). This study suggests that the spread of BoNT/A within the CNS is relatively
small and the toxin transport to more distant sensory and motor regions is unlikely at small doses
applied. However, high BoNT/A doses might lead to the occurrence of BoNT/A-cleaved SNAP-25 in
contralateral sensory and motor regions, revealing a trans-synaptic traffic within commissural spinal
cord neurons [118].
An indirect effect of the toxin on neuronal activation in distant sensory regions has also been
described [
21
]. Orofacial formalin injection-evoked c-Fos expression was suppressed by BoNT/A in the
periaqueductal gray and locus coeruleus. The toxin did not alter c-Fos expression in other sensory
regions in the diencephalon related to the motivational and affective pain modalities, such as the
hypothalamus, paraventricular thalamic nucleus, and amygdala. This observation suggests an indirect
modulation of neuronal activation in the ascending sensory regions. However, BoNT/A might have a
smaller effect on other pain modalities related to motivational and affective pain processing.
Toxins 2019, 11, 459 14 of 26
Figure 1. Actions of BoNT/A along the pain pathway.
In the orofacial formalin test, BoNT/A injected into the whisker pad did not modulate the levels
of monoamines and their metabolites, not supporting their in the BoNT/A antinociceptive action
[119]. BoNT/A’s effect on ascending pain processing in humans has not been examined by functional
imaging studies. However, a recent study in neurogenic overactive bladder indicated effects of
BoNT/A on most brain sensory regions known to be involved in the sensation and process of urinary
urgency, thus indicating BoNT/A effects beyond the bladder [120].
5. An Overview of Clinical Evidence of BoNT/A Analgesic Efficacy
As we previously discussed, the analgesic effect of BoNT/A has been shown in the treatment of
chronic migraine, including medication-overuse headache and several localized neuropathic pain
syndromes—all of them pain conditions that cannot be adequately alleviated with conventional
analgesics. The pharmacotherapeutic options for the treatment of chronic migraine and neuropathic
pain are very limited, with certain antiepileptics and antidepressants [121,122], and the development
of analgesics with novel mechanisms of action has proven to be extremely challenging [123]. Since
the pathophysiology of pain in these syndromes is complex, involving the sensitization of primary
and secondary afferents and also the enhanced activation of glial cells and reduced activity of the
endogenous pain modulating mechanisms, the peculiar long-lasting actions of BoNT/A on multiple
levels of the pain transmission as described in detail above offer a unique treatment modality. In
comparison to other classical and non-classical analgesic drugs, the greatest advantage of BoNT/A
use is a sustained effect after a single application and low risk for adverse effects even upon repeated
administrations.
While observational clinical data suggested a potential analgesic effect and in vivo animal
experiments consistently demonstrated an anti-hyperalgesic effect in a variety of models (Table 2),
the first experimental human studies did not demonstrate an unequivocal analgesic effect. In initial
studies on healthy human volunteers, the pain thresholds to heat, cold, or electrical stimulation as
well as capsaicin-induced pain ratings were unchanged after subcutaneous or intradermal
application of BoNT/A [124–126]. While baseline heat and cold pain thresholds were consistently
unaltered in later studies as well, it was demonstrated that BoNT/A increased mechanical pain
Figure 1. Actions of BoNT/A along the pain pathway.
In the orofacial formalin test, BoNT/A injected into the whisker pad did not modulate the levels of
monoamines and their metabolites, not supporting their in the BoNT/A antinociceptive action [
119
].
BoNT/A’s effect on ascending pain processing in humans has not been examined by functional imaging
studies. However, a recent study in neurogenic overactive bladder indicated effects of BoNT/A on
most brain sensory regions known to be involved in the sensation and process of urinary urgency, thus
indicating BoNT/A effects beyond the bladder [120].

Toxins 2019,11, 459 14 of 24
5. An Overview of Clinical Evidence of BoNT/A Analgesic Efficacy
As we previously discussed, the analgesic effect of BoNT/A has been shown in the treatment
of chronic migraine, including medication-overuse headache and several localized neuropathic pain
syndromes—all of them pain conditions that cannot be adequately alleviated with conventional
analgesics. The pharmacotherapeutic options for the treatment of chronic migraine and neuropathic
pain are very limited, with certain antiepileptics and antidepressants [121,122], and the development
of analgesics with novel mechanisms of action has proven to be extremely challenging [
123
]. Since the
pathophysiology of pain in these syndromes is complex, involving the sensitization of primary and
secondary afferents and also the enhanced activation of glial cells and reduced activity of the endogenous
pain modulating mechanisms, the peculiar long-lasting actions of BoNT/A on multiple levels of the
pain transmission as described in detail above offer a unique treatment modality. In comparison to
other classical and non-classical analgesic drugs, the greatest advantage of BoNT/A use is a sustained
effect after a single application and low risk for adverse effects even upon repeated administrations.
While observational clinical data suggested a potential analgesic effect and
in vivo
animal
experiments consistently demonstrated an anti-hyperalgesic effect in a variety of models (Table 2),
the first experimental human studies did not demonstrate an unequivocal analgesic effect. In initial
studies on healthy human volunteers, the pain thresholds to heat, cold, or electrical stimulation as
well as capsaicin-induced pain ratings were unchanged after subcutaneous or intradermal application
of BoNT/A [
124
–
126
]. While baseline heat and cold pain thresholds were consistently unaltered in
later studies as well, it was demonstrated that BoNT/A increased mechanical pain thresholds [
69
] and
significantly alleviated capsaicin-induced pain and allodynia [
127
,
128
], mustard oil–induced pain,
and histamine-induced itch [
69
]. In all cases, a significant reduction in the neurogenic vasodilation
was also measured, suggesting a reduced release of neuropeptides from capsaicin-sensitive nerve
terminals. Importantly, the effect of BoNT/A on intradermally applied capsaicin-induced responses
was demonstrated both by intramuscular and subcutaneous delivery [127,128].
To date, the highest level of clinical evidence exists for the efficacy of BoNT/A in chronic migraine
supported by several RCTs and metaanalysis [
8
,
9
]. The approved delivery route is intramuscular, and
the dosing is 5 U per site at 31–39 precise anatomical locations of the head and neck at 12-week intervals.
It was the first and, up to now, it remains the only approved clinical use of BoNT/A for a pain condition.
Initial open-label studies also suggested that BoNT/A might be effective to reduce headache severity
in other primary headache types, but later data from RCTs could not confirm a significant reduction
of headache frequency or severity in either episodic migraines or tension-type headaches. The lack
of a more general efficacy in headaches is not clear yet, since BoNT/A could reverse the sensitization
of meningeal nociceptors in rats [
68
] and it was clearly effective in experimentally-induced acute
trigeminal pain in humans [
127
,
128
]. A recent Cochrane metaanalysis of 4 RCTs conducted for chronic
migraine found that the overall reduction of migraine days compared to placebo was -3.07 days. This
could be considered as a modest improvement of symptoms, but one has to appreciate that the safety
profile of BoNT/A was excellent based on data from 23 RCTs. There were few adverse effects, among
which the most relevant were ptosis, muscle weakness, and neck pain, which were mostly transient.
Discontinuation rates were very low, which also suggests an overall good tolerability. A postmarketing
observational study which followed patients for 108 weeks also strongly supports the long-term safety
of repeated BoNT/A treatments [
20
]. This is especially relevant since adherence to oral preventive
antimigraine drugs is estimated to be extremely low [129,130].
However, controversy also exists about the efficacy of BoNT/A in chronic migraine treatment.
A recent double-blind randomized controlled trial on 90 patients suffering from chronic migraine
with medication overuse did not show any benefit from BoNT/A (155 IU) in addition to medication
withdrawal regarding the reduction of headache days and improvement of patients’ quality of life in
comparison to the placebo group. This trial is methodologically different from most of the previous
studies on chronic migraine because the placebo-group was injected with the masking doses of BoNT/A

Toxins 2019,11, 459 15 of 24
(17.5 U) in the forehead to prevent facial wrinkling. The authors assume that the unblinding in previous
experiments likely could positively affect the modest therapeutic benefit of BoNT/A. [131].
Attempts were also made to identify predicting factors associated with a better response to
BoNT/A treatment. Jakubowski et al. established that the imploding and ocular headache was more
frequently reported by responders, while the majority of non-responders described their headache as
exploding [
132
,
133
]. The authors theorized that the difference in headache character could be derived
from the sensitization of a different population of nociceptors—extracranial in the case of responders,
and intracranial in the case of non-responders. Another group found that the plasma level of CGRP
was higher in responders compared to non-responders, and a decrease of CGRP by treatment could
also be demonstrated [
134
,
135
]. On the other hand, the presence of cutaneous allodynia did not prove
to be a predicting factor for the efficacy of BoNT/A, even though it is considered to reflect the presence
of central sensitization [
132
,
133
,
136
]. A recent study on Korean patients also suggested that longer
disease duration could be associated with a poorer response [137].
RCTs also showed significant analgesic efficacy of BoNT/A in several neuropathic pain conditions,
such as trigeminal, posttraumatic or postherpetic neuralgia [
11
,
138
,
139
]. The onset of clinical efficacy
could be detected at week 1 in most reported trials. The duration of effect of a single administration
was followed up to three months. Heat and cold sensation was not affected by neuropathic pain
patients either [
107
], confirming the selective action of BoNT/A. The evidence is convincing, especially
the remarkable efficacy in trigeminal neuralgia, although we have to add that the number of patients
included in these trials and the placebo responses were small. Data from a larger industry-sponsored
trial for postherpetic neuralgia did not show significant reduction of pain scores [
140
]. The most
relevant RCTs on neuropathic pain are compiled in Table 3.
Table 3. Randomized clinical trials of BoNT/A for neuropathic pain.
Pain
Condition
Number of
Participants
Dose and
Delivery Route Primary Outcome Reference
posttraumatic
neuralgia 129
5 U/site
max. 200 U
i.d.
pain rating 0–10 BoNT/A−1.9
placebo −0.3 [34]
posttraumatic
neuralgia 246
5 U/site
max. 300 U
i.d.
pain rating 0–10 BoNT/A−1.9
placebo −0.6 [107]
postherpetic
neuralgia 60
5 U/site
max. 200 U
s.c.
pain rating 0–10
BoNT/A−4.5
lidocaine −2.6
placebo −2.9
[141]
postherpetic
neuralgia 30
5 U/site
max. 100 U
s.c.
pain rating 0–10 BoNT/A−4.6
placebo −0.5 [142]
postherpetic
neuralgia 117
2.5 U/site
max. 200 U
i.d.
pain rating 0–10 BoNT/A−1.2
placebo −1.2 [140]
trigeminal
neuralgia 42 5 U/site, 75 U
i.d. or s.m. pain rating 0–10 BoNT/A−6.05
placebo −1.88 [143]
trigeminal
neuralgia 20 5U/site, 100 U
s.c.
pain rating (0–10)
frequency of
paroxysms/day
BoNT/A−6.5
placebo −0.3
BoNT/A−32.7
placebo −0.1
[144]
trigeminal
neuralgia 36 50 U s.c.
pain rating (0–10)
frequency of
paroxysms/day
BoNT/A−4.1
placebo −1.25
BoNT/A−22.0
placebo −9.81
[145]
trigeminal
neuralgia 80
20 sites
25 or 75U
i.d. or s.m.
pain rating 0–10
BoNT/A 25U
−
4.24
BoNT/A 75U −5.4
placebo −2.96
[146]
diabetic
neuropathy 18 4U/site, 50U pain rating 0–10 BoNT/A−2.53
placebo −0.53 [147]
14 patients had postherpetic neuralgia; 2or postsurgical; i.d. intradermal, s.c. subcutaneous, s.m. submucosal.

Toxins 2019,11, 459 16 of 24
Small but significant pain relief was demonstrated in RCTs for musculoskeletal pain, in particular
plantar fasciitis, tennis elbow, and low back pain. The efficacy could not be conclusively proven
in patients with myofascial pain syndromes [
11
,
148
]. In the case of osteoarthritis, some trials have
demonstrated efficacy for patients with refractory osteoarthritic pain. Results for RCTs for osteoarthritic
pain are summarized in Table 4.
Table 4. Randomized clinical trials of BoNT/A for low back pain and osteoarthritic pain.
Pain Condition Number of
Participants
Dose and
Delivery Route Primary Outcome Reference
low back pain 31 40 U/site
200 U i.m.
% of responders
(50% reduction in
pain rating)
BoNT/A 73.3%
placebo 25% [149]
refractory shoulder
pain 36 100 U i.a. pain rating 0–10 BoNT/A -2.4
placebo -0.8 [150]
refractory painful
total knee
arthroplasty
54 100 U i.a.
% of responders
(2-point reduction
in pain ratings)
BoNT/A 71%
placebo 35% [151]
knee osteoarthritis 176 200 U or 400 U
i.a. pain rating 0–10
BoNT/A 200 U -1.6
BoNT/A 400U -2.1
placebo -2.1
[152]
knee osteoarthritis 121 200 U i.a. pain rating 0–10 BoNT/A -2.2
placebo -2.5 [153,154]
i.m. intramuscular, i.a. intraarticular.
Because of the lack of clear guidelines regarding the amount of BoNT/A injected, the route and
site of injection, and the number of injections, clinical studies vary in treatment protocols, which make
them difficult to compare. Thus, future clinical trials should be more standardized regarding study
design, patients’ stratification, measurement outcomes, and study endpoints, and those studies should
look for both short- and long-term outcomes relevant for BoNT/A efficacy, tolerability, and safety.
6. Conclusions
The success of BoNT/A therapy in treating certain chronic pain conditions and individual patients
may be influenced by a variety of factors related to its main mechanisms of action. In the sensory system,
BoNT/A action may be restricted to certain neuronal populations mediating pain hypersensitivity,
which could explain its efficacy only in some types of chronic pain or patient subpopulations. Longevity
of action of BoNT/A is most likely to be related to its cellular localization, which enables a long-term
effect after a single application. Further characterization of BoNT/A effects on multiple sites of action
on its way from the periphery to CNS are the next necessary steps to explain its antinociceptive effect
and help to improve its clinical use.
Author Contributions:
Conceptualization, I.M.; Writing—original draft preparation, I.M., L.B.-R. and K.B.;
Writing—review and editing, Z.H., K.B., I.M. and L.B.-R.; Funding acquisition, Z.H.
Funding:
The present study was supported by Hungarian Grants: National Brain Research Program B
(KTIA_NAP_13-2014-0022), EFOP-3.6.1.-16-2016-0004, GINOP 2.3.2-15-2016-00050 “PEPSYS”. Experimental
work of L.B. and I.M. was supported by Croatian Science Foundation (IP-2014-09-4503).
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

Toxins 2019,11, 459 17 of 24
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