What is Botox?

By Qinyi Wang and Kah Yan Ng

Imagine a poison so lethal that it can wipe out the entire population of London with just enough toxin to cover a 2 pence coin. If dispersed evenly, a single gram of this substance in crystalline form can kill one million people. It is the most potent toxin in the world, natural or synthetic, with a lethal dose of only 1.3–2.1 ng/kg in humans (Arnon et al. 2001). The most obvious application of something so potent is in the development of weaponry for warfare, though human ingenuity has far exceeded such mundane constrictions of imagination. What if I told you, that this ‘poison’ is commonly administered as a beautifying treatment in cosmetic procedures?

What most people refer to as ‘botox’ is an extremely diluted version of the botulinum toxin produced by Clostridium botulinum (C. botulinum). These are a group of gram-positive, anaerobic bacteria prevalent in marine sediments and soil worldwide. All 4 currently recognised groups of C. botulinum (I-IV) produce botulinum toxin. They usually lie dormant and harmless in the form of spores, and the real danger presents once they develop into active bacteria and produce botulinum, a powerful neurotoxin. This then leads to the condition known as botulism, which if untreated, results in the death of the patient.

The most common form of botulism is food-borne botulism, though other forms exist. In food-borne botulism, the toxin crosses the intestinal epithelial barrier into the lymphatic system after ingestion and reaches the systemic circulation where it acts on peripheral cholinergic nerve terminals. The pathways in which the botulinum acts are universal once it gains systemic access with an exception of reaching the central nervous system as the toxin is unable to cross the blood-brain barrier. (Restani et al., 2012)

So how exactly does this neurotoxin cause muscular paralysis? It boils down to the peripheral cholinergic synapses which use acetylcholine as neurotransmitters. During calcium influx into the pre-synaptic neuron, acetylcholine is released into the synapse to trigger a muscle response. The exocytosis of acetylcholine is inhibited by the binding of botulinum toxin, paralysing the muscles that control action (Lawrence, 1998).

There are 3 steps to this mechanism: binding, internalisation and a final step of inhibition (Melling, Hambleton and Shone, 1988). First, the toxin exhibits ability for dual binding where it binds to a protein receptor called synaptotagmin and a polysialoganglioside receptor. This binding is selective, irreversible and rapid due to dipole-dipole attraction. Botulinum toxin is a positive dipole which has high affinity for the anionic receptors. Once bound, the toxin-receptor complex formed is engulfed via endocytosis into the cell. (Nigam and Nigam, 2010)

The onset of muscle paralysis is slow as the toxin takes effect only after 24 – 72 hours. (Nigam and Nigam, 2010). As the binding step is rapid, this suggests that binding is not the limiting factor. A previous study performed on synaptosomes from rodents’ brains showed that radioactively labelled toxins were inhibited by an excess of unlabelled toxins, suggesting competitive inhibition as a result of limited binding sites (WILLIAMS et al., 1983).

Binding itself is not enough for an inhibition of acetylcholine release to take place. For the toxin to access proteins that facilitate neurotransmitter release, internalisation into the cytosol across the presynaptic membrane is required. This process involves the generation of the L chain through a cleavage of the toxin molecule. ATPase proton pump causes a pH gradient across the membrane, driving the translocation of L chain into the cytoplasm as evidenced by an experiment where ATPase

inhibitors completely negated the intoxication effects of the toxin on nerve terminals (Rossetto, Pirazzini and Montecucco, 2014).

Upon reaching the cytosol, the L chain acts as an enzyme and cleaves a SNAP receptor known as SNARE. This receptor localises in the cytosol and is required for the release of vesicles with acetylcholine into the synapse. When SNARE is cleaved, the release of acetylcholine is inhibited (Lawrence, 1998)

Given that the primary effect of the botulinum toxin is paralysing muscles through acting on the cholinergic synapses, this makes for some interesting applications. What is perhaps the most ‘well-known’ application is the Botox injection, where individuals can voluntarily have a commercial form of botulinum injected into their body. Of the 8 forms of botulinum toxin, serotype A is approved for human use. Intra-muscular injection of the toxin can reduce the appearance of wrinkles, which therefore makes facial muscles the favoured target. Wrinkles are formed through dermal atrophy and repetitive contraction of underlying muscles. Localised delivery of botulinum toxin stops muscles from contracting, smoothing the skin as overactive muscles relax. The effect of each injection lasts 3-4 months, and another dose can be administered to maintain the effects. (Camargo et al., 2014)

Apart from cosmetic procedures, botulinum can be used to treat neurological disorders such as dystonias. Dystonia is characterised by abnormal muscle tone and muscular spasms, resulting in involuntary twisting movements and unnatural postures. Injecting botulinum intramuscularly can weaken dystonic muscles and ameliorate symptoms, and are thus used to treat cervical, oromandibular, lingual dystonia and blepharospasm (Cloud and Jinnah, 2010). Additionally, botulinum has also been shown to inhibit cholinergic autonomic innervation of glands and smooth muscle. It can therefore be used in treating conditions such as hypersecretory diseases, smooth muscle pain and spasms, and so on (Cooper, 2007).

Due to the acceptance of botulinum as a treatment and stringent regulations behind the production of injections, there are very few casualties in authorised clinics. There are, however, side effects, which usually only manifests as temporary paralysis in surrounding musculature. In some rare cases, the toxin may spread throughout the body with large doses of botulinum toxin and lead to generalised muscle weakness, urinary incontinence, respiratory difficulties, etc. (Yiannakopoulou, 2015). Repeated use can lead to the development of neutralising antibodies against the toxin, making it ineffective (Fabbri et al., 2016). Thus, lowest effective doses should be calculated individually for each patient and injections should be performed at ≥3-month intervals (Cloud and Jinnah, 2010).

The application of this toxin in cosmetic procedures and in some neurological therapy is largely accepted. However, more studies have to be conducted to elucidate the mechanism for systemic toxin spread in order to minimise severe side effects, especially in long-term usage. Moreover, with the discovery of many different types of botulinum neurotoxins, establishing which ones have increased potency and durations of action could point to novel therapeutic toxins that benefit more people.

References:

Arnon SS, et al. (2001) ‘Botulinum toxin as a biological weapon: medical and public health management’, JAMA. 2001(8), pp.1059-70. doi: 10.1001/jama.285.8.1059.

Camargo, C. P. et al. (2014) ‘Botulinum toxin for facial wrinkles’, Cochrane Database of Systematic

Reviews, 2014(9). doi: 10.1002/14651858.CD011301.

Cloud, L. J. and Jinnah, H. (2010) ‘Treatment strategies for dystonia’, Expert Opinion on Pharmacotherapy, 11(1), pp. 5–15. doi: 10.1517/14656560903426171.

Cooper, G. (2007) ‘Therapeutic uses of botulinum toxin’, Therapeutic Uses of Botulinum Toxin, 5(1), pp. 1–238. doi: 10.1007/978-1-59745-247-2.

Fabbri, M. et al. (2016) ‘Neutralizing Antibody and Botulinum Toxin Therapy: A Systematic Review and Meta-analysis’, Neurotoxicity Research. Springer US, 29(1), pp. 105–117. doi: 10.1007/s12640-015-9565-5.

Lawrence, E. (1998) ‘Getting to grips with botulinum toxin’, Nature, 3(April). doi: 10.1038/news981029-2.

Melling, J., Hambleton, P. and Shone, C. C. (1988) ‘Clostridium botulinum toxins: Nature and preparation for clinical use’, Eye (Basingstoke). Nature Publishing Group, 2(1), pp. 16–23. doi: 10.1038/eye.1988.5.

Nigam, P. and Nigam, A. (2010) ‘Botulinum toxin’, Indian Journal of Dermatology, 55(1), p. 8. doi: 10.4103/0019-5154.60343.

Restani, L. et al. (2012) ‘Botulinum Neurotoxins A and E Undergo Retrograde Axonal Transport in Primary Motor Neurons’, PLoS Pathogens, 8(12). doi: 10.1371/journal.ppat.1003087.

Rossetto, O., Pirazzini, M. and Montecucco, C. (2014) ‘Botulinum neurotoxins: Genetic, structural and mechanistic insights’, Nature Reviews Microbiology. Nature Publishing Group, 12(8), pp. 535–549. doi: 10.1038/nrmicro3295.

WILLIAMS, R. S. et al. (1983) ‘Radioiodination of Botulinum Neurotoxin Type A with Retention of Biological Activity and Its Binding to Brain Synaptosomes’, European Journal of Biochemistry, 131(2), pp. 437–445. doi: 10.1111/j.1432-1033.1983.tb07282.x.

Yiannakopoulou, E. (2015) ‘Serious and long-term adverse events associated with the therapeutic and cosmetic use of botulinum toxin’, Pharmacology, 95, pp. 65–69. doi: 10.1159/000370245.