Amy Malhowski
Biology 360
March 30, 2005
Figure taken from: http://www.consultingroom.com/Aesthetics/Products/Product_Display.asp?ID=1

Bioterrorism!
Figures taken from: http://www.safetycentral.com/bottoxfacin.html
http://archives.cnn.com/2002/ALLPOLITICS/06/12/bush.terror/

Figure taken from: http://lingua.utdallas.edu:7000/1442/

•
Historical background of C. botulinum
• Transmission of Botulinum toxin
• Molecular pathogenesis
• Therapeutic uses of Botulinum toxin
• Concluding remarks

• Flaccid paralysis of muscles
– by toxin from
Clostridium botulinum
• Three types – via route of entry of bacteria
– Foodborne, infant, wound
• Mainly foodborne outbreaks
• Now bioterrorism threat

• “botulism” from botulus (sausage) from outbreak of consuming improperly cooked sausage
• Published 1 st case studies on botulinum intoxication
• Accurately described neurological symptoms
• 1 st to propose therapeutic use of toxin
Figure adapted from: Erbguth, 2004.

Figure taken from Caya, et al.
, 2004.

• Emile Pierre van Ermengem (1895)
– 1 st to connect botulism to bacterium from raw, salted pork & postmortem tissues of botulism victims
– Isolated bacterium, naming it Bacillus botulinus

• Strict anaerobe
• Gram-positive
• Bacillus (rod) shape
• Ubiquitous in terrestrial environment
• Virulence factor = Botulinum toxin
– Released under specific conditions
Figure taken from http://www.jhsph.edu/Publications/Special/cover2.htm

• Great potential in toxicity
• Toxin tested as bioweapon during WWII
– aborted when toxin did not affect test animals
(donkeys)
• BoNT no longer considered good bioweapon

• Fort Detrick (1946) – bioweapon research –
1 st time mass produce toxin
• Nixon terminates all research on biowarfare agents
(1972)
• Schantz produces batch 79-11 (1979)
– used until 1997
• Batches made in 1991
– Botox® by Allergan Inc., Irvine, CA

• Bioterrorism/outbreaks
• Kerner – use in therapeutics
• Recently – BoNT as therapeutic agent for neuromuscular disorders

• Historical background of
C. botulinum
•
Transmission of Botulinum toxin
• Molecular pathogenesis
• Therapeutic uses of Botulinum toxin
• Impediments in Treatment
• Concluding remarks

• Mostly via improperly cooked food
• Conditions to produce toxin not completely understood
• Complex route of transmission
– Ingestion/injection
– Progenitor toxin complex
– Absorbed into tissue  circulated in blood
– Dock onto receptors of neuron  transcytosis
 binds up acetylcholine
 paralysis

• Seven different subtypes of botulinum toxin
– A, B, C1, D, E, F, and G
• Same general mechanism for muscular paralysis
• Vary in structure, target site, & toxicity
• Only two manufactured for commercial use
– A and B

Serotype
A
B
C1
D
E
F
Cellular Substrate
SNAP-25
VAMP/Synaptobrevin
Cellubrevin
Syntaxin 1A, 1B
SNAP-25
VAMP/Synaptobrevin (18, 181)
Cellubrevin (181)
SNAP-25(46)
VAMP/Synaptobrevin (181, 182)
Cellubrevin
VAMP/Synaptobrevin
Target Cleavage Site
Gln197-Arg198
Gln76-Phe77
Gln59-Phe60?
Lys253-Ala254
Lys252-253
Lys59-Leu60
Ala67-Asp68
Lys42-Leu43?
Arg180-Ile181
Gln58-Lys59
Gln41-Lys42?
Ala81-Ala82 G
Figure adapted from: Aoki. 2004. Curr Med Chem. 11: 3085-3092.

• Historical background of
C. botulinum
• Transmission of Botulinum toxin
•
Molecular pathogenesis
• Therapeutic uses of Botulinum toxin
• Impediments in Treatment
• Concluding remarks

BoNT synthesized as single-chain polypeptide
(inactive form)
Polypeptide cleaved by protease to create dichain structure (active form)
BoNT binds to epithelium, transcytosed, reaches general circulation
Receptor-mediated endocytosis at peripheral cholinergic nerve endings
In cytosol, toxin cleaves target, blocking neurotransmitter release = flaccid paralysis

Figure taken from: Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.

5’ ha operon ha70 ha17 ha34
RNAP
Core
BotR/A botR/A v ntnh
ntnh-bont/A operon bont/A
3’
Figure adapted from: Raffestin, S., et al., 2005. Molec. Microbiol. 55: 235-249.

Aoki. 2004. Curr Med Chem. 11: 3085-3092.
Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.

Figure taken from:
Arnon, et al.
2001.

• Bioterrorism agent – Category A
• Local paralytic agent – Botox®
• Therapeutic agent
– Neuromuscular disorders
– Pain management

• Use Botulinum toxin type A (Botox®)
• Many cosmetic uses
• Few clinical side effects
• Fast acting – 6 hours post injection
• Effects last 3-6 months
• Serial injections required to maintain results

• Local effects = dose dependent
• Injection site affects physical outcome
Before Botox® After Botox®
Figures adapted from: Mendez-Eastman. 2003. Plast. Surg. Nurs. 23: 64-70.

• Historical background of
C. botulinum
• Transmission of Botulinum toxin
• Molecular pathogenesis
•
Therapeutic uses of Botulinum toxin
• Impediments in Treatment
• Concluding remarks

• Botox® used in aesthetics
 therapeutic use in neuromuscular disorders
• BoNT/A = Botox® - Allergan, Inc.
• BoNT/B = MYOBLOC™ - Elan Pharmaceuticals

• Purified BoNT/A = Botox®
• Treat medical conditions characterized by muscle hyperactivity/spasm
– blepharospasm, strabismus, cervical dystonia, glabellar lines, spastic dystonia, limb spasticity, tremors, chronic anal fissure, hyperhidrosis, etc.
• Currently only FDA approved for 4 disorders
– Blepharospasm (focal dystonia)
– Strabismus
– Cervical dystonia
– Hyperhidrosis

• CD – involuntary contractions of neck and shoulder muscles
• FDA approved injections with BoNT/A (2000)
• BoNT/A injected into affected muscles to reduce muscle contraction
• BoNT/A effectively reduces muscle spasticity and pain associated with CD

• Phase 3 randomized, multi-center, double blind, placebo-controlled study on treatment of CD with
Botox ® (1998)
• 170 subjects (88 in Botox® group, 82 in placebo group), analyzed until 10 wks post-injection
• Study suggests majority of patients had beneficial response by 6 th week

• Multicenter, double-blind, randomized, controlled trial with
Dysport® to treat CD in the USA (2005)
• Patients (80) randomly assigned to receive Dysport® (500U) or placebo
• Dysport® significantly more effective than placebo at weeks
4, 8, and 12
• Dysport® group had 38% with positive treatment response, with median duration of response of 18.5 weeks

• BoNT use in controlling pain-associated disorders
• Data suggests BoNT acts in complex manner – not just controlling overactive muscle
• BoNT inhibits the release of neurotransmitters
(glutamate and substance P) involved in pain transmission

Figure taken from: Aoki, 2003.

Figure taken from: Aoki, 2003.

• Acute pain (phase 1) - not relieved by BoNT/A
• Inflammatory pain (phase II) - relieved by BoNT/A
• Increasing doses decrease phase II pain appreciably
• Antinociceptive activity maintained longer with higher dose of BoNT/A
Figure taken from: Aoki, 2003.

• Formalin challenge 5 days post-injection with BoNT
 dose-dependent decrease in Glut release
• BoNT/A prevents increase of formalin-induced Glut release
Figure taken from: Aoki, 2003.

A) Antinociceptive
Activity of BoNT/A in formalin-challenged rats.
B) Subcutaneous BoNT/A injection reduces formalin-induced glutamate release in rat paw in a formalin-challenged inflammatory pain animal model.
Figures taken from: Aoki, 2003.

• BoNT mechanism = specific
• Uses are diverse
– Local flaccid paralysis
– Reducing muscle spasticity
– Reducing pain
• Currently use of BoNT
– muscle disorders and associated pain

• Historical background of
C. botulinum
• Transmission of Botulinum toxin
• Molecular pathogenesis
• Therapeutic uses of Botulinum toxin
•
Impediments in Treatment
• Concluding remarks

• FDA approval pending for many disorders
• Fleeting effects – need repeated injections
• Socioeconomics – less expensive than surgery
BUT not permanent
• Social constraints –
– More research needed
– stigma in using deadly toxin for good use

• Toxin = great therapeutic agent!
• Research needed to understand mechanism of release of BoNT from C. botulinum
• Few impediments in therapeutics
• Future with Botox® is bright!

• Chris White-Ziegler
• My readers: Caitlin Reed & Natalia Grob
• Bio 360 students
Figure taken from: http://www.jwolfe.clara.net/Humour/MedMiscel.htm

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