National Botulinum Research Center, and Department of Chemistry and Biochemistry,

University of Massachusetts Dartmouth, 285 Old Westport Road, Dartmouth, MA 02747, USA

Phone: 508-999-8588

Fax: 508-999-8451

Email: bsingh@umassd.edu

Summary

Botulinum neurotoxin is produced by the anaerobic bacterium Clostridium botulinum. It is the most poisonous poison known. Very small amounts of botulinum toxin can lead to botulism, a descending paralysis with prominent bulbar symptoms and often affecting the autonomic nervous system. Because of its extreme toxicity, it is placed in Category A list of biothreat agents. Previously known only as a cause of a serious and often fatal paralysis acquired through ingestion of contaminated food, the toxin recently has been used for medicinal and cosmetic purpose. The toxin causes paralysis by blocking the presynaptic release of acetylcholine at the neuromuscular junction. Botulinum toxin A was first approved by US Food and Drug Administration in the year 1989, to treat two eye muscle disorders (blepharospasm and strabismus) and in the year 2000 to treat cervical dystonia, a neurological movement disorder causing severe neck and shoulder contractions. The use of botulinum toxin has further expanded and now it is being used as a safe and effective way to treat several neuromuscular disorders and to reduce facial wrinkles without surgery. Research is underway to use botulinum toxin for drug delivery, migraine, Alzheimer and treatment of cancer. Its effectiveness in the treatment of various neuromuscular diseases and its multi therapeutic uses has placed the toxin on top of new drug development list.

Journey of botulinum neurotoxins from food-poisoning to neuromedicine

Although botulism occurs rarely, it is dangerous because of its high fatality rate.åÊ Clinical descriptions of botulism possibly reach as far back in history as ancient Rome and Greece.åÊ However, the relationship between contaminated food and botulism was not defined until the late 1700s. In 1793, the German physician, Justinius Kerner, deduced that a substance in spoiled sausages, which he called Wurstgift (German for sausage poison), caused botulism.åÊ The toxin’s origin and identity remained elusive until Emile von Ermengem, a Belgian professor, isolated Clostridium botulinum, (Clostrodium from Latin Kloster meaning spindle, and botulinum from Latin botulus, meaning sausage) in 1895 and identified it as the source of the deadliest poison known¹.

Botulinum toxins (BoNTs) are the most toxic compounds known, with an estimated toxic dose (serotype A) of only 0.001 micrograms/kg of body weight; botulinum toxin is 15,000 times more toxic than the nerve agent VX and 100,000 times more toxic than sarin. On a molar basis, they are 300-fold more lethal than diphtheria toxin, 3ÌÑ10⁴ more toxic than ricin, 1ÌÑ10⁹ more toxic than curare and 1ÌÑ10¹¹ more toxic than sodium cyanide².

Botulism is one of the most terrifying paralyzing diseases to afflict humankind. The clinical spectrum of botulism continues to expand and is today divided into five clinical categories³:åÊ (i) Food-borne botulism results from ingestion of food containing pre-formed neurotoxin, produced by clostridial organisms that contaminate inadequately processed food; (ii) Infant botulism is caused by the ingestion of spores that then germinate and produce toxin in the infant’s gastrointestinal tract; (iii) Wound botulism arises as a consequence of toxin produced in wounds contaminated with the clostridial bacterium; (iv) Hidden botulism is an adult variation of infant botulism, (v) Inadvertent botulism which is an unintended consequence of treatment with botulinum toxin A. A sixth category of intentional botulism refers to botulism caused by intentional contamination of food or environment.

Clostridial neurotoxins, botulinum and its cousin, tetanus neurotoxin (produced by Clostridium tetani) act as metalloproteinases that enter peripheral cholinergic nerve terminals and cleave proteins that are crucial components of the neuroexocytosis apparatus, causing a persistent but reversible inhibition of neurotransmitter release. While tetanus toxin affects interneurons in the spinal cord, botulinum toxin affects peripheral cholinergic transmission^4,5.

Because of their extreme toxicity, ease of production, and robust stability under adverse environmental conditions, BoNTs are on the top of the list of biological warfare threats, and have been listed as Class A bioterror agents by the US Centers for Disease Control (CDC). The toxin produced in its native form (consisting of the neuroxin and five neurotoxin associated proteins or NAPs) can survive in open environment for months, and can also survive the digestive proteases in the gastro-intestinal tract. NAPs play a critical role in this protection, and seem to have evolved with the neurotoxin for this purpose. Although BoNTs have not been used successfully as a bioweapon, attempts have been made in the past⁶. Japan’s Arsinio group made an unsuccessful attempt in the 1980s, and UNSCOM discovered in 1991 that Iraq had loaded 10,000 liters of a 19,000 liter stock of botulinum agent in its scud missiles. While the 10,000 liters were destroyed at that time, the remaining 9,000 liters were considered as suspected weapons of mass destruction that were unaccounted for, and formed, in part, the basis of the U.S. government’s 2003 invasion of Iraq.

Paradoxically, because of their extreme neurospecificity, BoNTs are being exploited in the treatment of a myriad of neuromuscular disorders and for the removal of facial wrinkles.

The biothreat

Botulinum neurotoxins are the only toxin agent, in contrast to the organism, in Class A list of biothreat. This situation requires extraordinary measures for its detection since this is the most toxic agent known to mankind today. Challenges include detection of extremely low quantities (in picograms), and identification of specific toxin types. In addition, one must also evaluate if the toxin is active or inactive, since being a protein it is labile to environmental and biological inactivation. One also needs to detect it fast as once the symptoms are developed (within 24-48 hours of exposure); it becomes too late for any remediation course. The toxin strategically blocks the release of acetylcholine neurotransmitter at the nerve-muscle junctions, causing flaccid muscle paralysis. This results in breathing problems, leading to ultimate death if artificial ventilation is not available. The problem is further aggravated because of the long lasting paralysis, thus necessitating long-term (weeks to months) intensive care unit (ICU) surveillance and care. It is estimated that currently there are only about 2,000 civilian ICUs in the United States. So, even a mild bioterror attack can result in a major disaster, as it will not only concern the patients who are exposed to the toxin but also all those others who need to use ICUs for short terms.

While no successful botulinum bioterror incident has occurred in the world so far, a mathematical model of a cows-to-consumers supply chain associated with a single milk-processing facility that is the victim of a deliberate release of botulinum toxin has been carried out7. Because centralized storage and processing lead to substantial dilution of the toxin, a minimum amount of toxin is required for the release to do damage. Irreducible uncertainties regarding the dose-response curve prevented from quantifying the minimum effective release. However, if terrorists can obtain enough toxin, and this may well be possible, then rapid distribution and consumption result in several hundred thousand poisoned individuals if detection from early symptomatics is not timely. Timely and specific in-process testing has the potential to eliminate the threat. Investigation of improving the toxin inactivation rate of heat pasteurization without sacrificing taste or nutrition is warranted.

In addition, certain serotypes of BoNT are known to cause botulism in horses, cattle, birds, and fish. It is theoretically possible to target industries related to these animals for bioterror, which may become environmental and business disasters.

Current antidotes available are equine antibodies raised against different serotypes of botulinum neurotoxin. The US government has recently contracted a Canadian company to prepare equine antitoxins against all the BoNT serotypes. However, this antitoxin has very limited window of opportunity for treatment. This is because the antitoxin can work only against toxins which have not entered the nerve cells. Once the toxin has entered the nerve cells, it causes the chemical changes inside the cells which lead to muscle paralysis. Currently, there is no drug to rescue poisoned nerve cells, although many efforts are underway, with little success. This failure has many reasons, but most important is the tricky characteristics of the toxin itself which may have evolved over billions of years.

One easy way to tackle diseases like botulism could be to vaccinate the population against the toxin, similar to what is currently done against tetanus. While an effective vaccine is currently not available, it is not that difficult to develop one. Efficacy of the current vaccine available from CDC is not guaranteed, as it was prepared in the 1970s, and is approved by the FDA as just an investigational new drug (IND) for use by only researchers in the field. The major reason this vaccine has not been developed is the fact that once vaccinated the person becomes ineligible for treatment with the toxin for many of the neuromuscular disorders. Research is needed to reconcile these two different needs.

The Botulinum Research Center at UMass Dartmouth is working with scientists from academia (Tufts, Harvard, MIT Lincoln Lab, University of Wisconsin, University of Missouri, New York University, UMass Medical School, etc.), government labs (DoD labs, CDC, FDA, USDA, Sandia National Lab, Brookhaven National Lab, et al.), and Industry (Newton Photonics, Microbiotix, Radix Biosolutions, IPSEN ‰ÛÒ UK, MERZ-Germany, etc al.) to develop biosensors, antidotes, vaccine, etc. to mitigate the biothreat; as well as improved biomedical product for therapeutic uses. The center holds workshops to train research personnel and medical staff to deal with the toxin whether for medical use or for countering the biothreat.

Brief history of the medicinal use

The extreme toxicity and its exclusive action on neuromuscular junctions led to the investigation of its medicinal use for neuromuscular disorders. Researches have envisioned the use of the deadliest toxin for therapeutic purpose. The first preparation of crystalline forms of BoNT/A toxin goes back to World War II when an American researcher named Dr. Edward Schantz isolated the crude form of the toxin.åÊ Purification of the crude form of botulinum type A complex was carried out from1940 to 1960 at Fort Detrick by Dr. Edward Schantz and his colleagues⁸.åÊ The use of botulinum toxin type A as an injectable selective muscle-weakening agent was investigated experimentally in monkeys in the 1960s and late 1970s and in humans in the late 1970s by Alan Scott, a physician in California, who used it to treat strabismus patients.

Subsequently, in the 1980s, BoNT/A was used for blepharospasm and other focal dystonias^8-12. The United States Food and Drug Administration approved BoNT/A (BOTOXå¨, Allergan, Inc.) in the management of strabismus, blepharospasm and related facial spasms in 1989 and cervical dystonias in 2000. Botulinum toxin type B first became available commercially in the USA in 2000, and was approved for cervical dystonia, but not for blepharospasm or strabismus. Botulinum toxin A is manufactured under the trade name BOTOXå¨ (Allergan, Inc.) in the United States and DYSPORTå¨ (Ipsen Ltd.) in Europe. In the last few years, BoNT/A has also been produced in China. The Chinese toxin (Lanzhou Biological Products Institute, China) is being marketed in Asia as BTX-A. Botulinum toxin B is available in the U.S. as MYOBLOCå¨ and in Europe as NEUROBLOCå¨ (Solstice, Inc.)^13-15. A new product based on the purified BoNT/A is now available as Xeominå¨ from the Merz Company in Germany.

Botulinum toxin A has been successfully used for the treatment of many disorders related to excessive muscle contraction, such as strabismus, blepharospasm, hemifacial spasm and cervical dystonia. BoNT-A also significantly reduces pain associated with craniocervical dystonia‰Û҉ÛÒan effect that has long been considered secondary to its muscle relaxant action. Botulinum toxin A also has been used successfully to treat several different types of headaches, including tension type headaches, cervicogenic headaches and migraine. Botulinum toxin is now widely considered as a pharmaceutical agent with multiple uses, and has propelled into the public eye after it was widely reported to act as an anti-wrinkle drug for facial cosmetic enhancement. This has established its new image as a glamour drug. Botulinum toxin is reported to be useful in more than 50 conditions, with indications spanning many specialties. In the late 1980s, a group of physicians at Columbia University noticed that patients who received injections for nerve disorders also experienced cosmetic improvement. They pursued this investigation and soon developed the botulinum toxin therapy in common use today.

Table: Recent medicinal use of botulinum neurotoxin16.
Disorders	Dysfunction
1. Chronic headache	Tension-type headache50
2. Overactive bladder (OAB)	Spastically contraction of detrusor bladder muscles51
3. Laryngeal dystonia	Vocal cord dystonia52
4. Temporomandibular joint dislocation	Dysfunction of the joint, muscles of the jaw53
5. Dysuria or urinary retention	Non-relaxing urethral sphincter54
6. Tourette’s syndrome	Involuntary, rapid, sudden movements of muscles5554
7. Focal hyperhidrosis	Excessive sweating56

Still, there is much we do not know about botulinum neurotoxin, and we are in the phase where its use as a diagnostic and therapeutic agent is in much demand.åÊ Areas of investigation include long-term effects, optimal treatment regimens, and reasons for treatment failure.åÊ Nevertheless, many efforts have been made to understand its structure, function and biochemical properties and pharmacological actions to improve its therapeutic uses for various neuromuscular disorders.

Botulinum, a minefield for intricate science knowledge

Genetically, botulinum neurotoxins are produced as part of a group of genes which has a common regulatory factor for the expression of the gene cluster as a whole. The proteins collectively form a stable complex, which is critically important to their role in food poisoning and to the bioweapon role of this agent. This complex is also used as the therapeutic product because of its naturally stable formulation. Overall, these topics provide an important coalescence of interest in fundamental research, applied technology development and major business and health concerns.

Consequently, botulinum neurotoxins have become a tremendous target of research and subsequent publications within the past 25 years¹⁷. A dramatic enhancement of botulinum-related publications has been noted in the United States, particularly since the mid-1990s. An obvious connection of this enhancement in publications is the approval of botulinum neurotoxin as a therapeutic drug against several neuromuscular disorders. Data mining exercises of Pubmed, a service of the U.S. National Library of Medicine and the U.S. National Institutes of Health suggested that while articles referring to food poison and detection were predominant in the 1970s and early 1980s, mode, mechanism and receptor studies dominated the 1990s. Research with therapeutic products like Botoxå¨ and Dysportå¨ started to increase in the mid 1990s; in 2007 about half of the articles were related to therapeutic products. A small number of botulinum articles have started appearing since 2000 with terms like “bioweapon” and “antidotes”. This wide range of topics related to botulinum provides a strong base of scholarship in the field, and a new journal, The Botulinum Journal (TBJ), was launched in 2008.

Concluding remarks

Generally, botulinum neurotoxins are considered lethal to humans when used in high doses either by deliberate or accidental exposure. Because of the tiny amount needed to cause mortality and morbidity, and because currently there is no drug to rescue the poisoned nerves, BoNTs are capable of causing a major disaster to the healthcare industry if used intentionally. Botulinum toxin is also a rare example of a potentially lethal biological agent that can be used as a medicine for several neuromuscular disorders. It has many unique characteristics including exclusive substrate specificity and localized action. Despite its extreme toxicity, it has been widely used as a wrinkle remover which is perhaps the single most popular use. Although it is considered the most toxic substance known to humankind, researches are turning this killer into cure, and the days may not be too far when it will be referred to as botulinum neuromedicine (BoNEM) rather than botulinum neurotoxin.

Note: Bal Ram Singh is the founding director of the National Botulinum Research Center at UMass Dartmouth. The Center holds annual symposium on botulinum research, which attracts national and international researchers from academia, industry, and government laboratories and agencies. Preparation of this article was in part supported by funds from DARPA (W911NF-07-1-0623).

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