Botulinum toxin type A: basic pharmacological profile and therapeutic applications

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.


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 nonhemagglutinin (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). 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 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). 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).
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, longlasting 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).

Pain models Results
Inflammatory pain models: 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). 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 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 nonpolar 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.