Transcript Clostridium botulinum Toxin: Friend or Foe?

Clostridium botulinum Toxin:
The Neuromuscular Wonder Drug
Amy Malhowski
Biology 360
March 30, 2005
Figure taken from: http://www.consultingroom.com/Aesthetics/Products/Product_Display.asp?ID=1
Public Perception of Botulinum Toxin
Bioterrorism!
Figures taken from: http://www.safetycentral.com/bottoxfacin.html
http://archives.cnn.com/2002/ALLPOLITICS/06/12/bush.terror/
And of course…Botox®
Aka “The Fountain of Youth”
Figure taken from: http://lingua.utdallas.edu:7000/1442/
Outline of Talk
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Historical background of C. botulinum
Transmission of Botulinum toxin
Molecular pathogenesis
Therapeutic uses of Botulinum toxin
Concluding remarks
What is Botulism?
• 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
The History of Botulinum Toxin
• “botulism” from botulus (sausage)
from outbreak of consuming
improperly cooked sausage
• Published 1st case studies on
botulinum intoxication
• Accurately described neurological
symptoms
• 1st to propose therapeutic use of toxin
Figure adapted from: Erbguth, 2004.
Symptoms of Botulism
Figure taken from Caya, et al., 2004.
Finding the Culprit
• Emile Pierre van Ermengem (1895)
– 1st to connect botulism to bacterium from raw,
salted pork & postmortem tissues of botulism
victims
– Isolated bacterium, naming it Bacillus botulinus
Clostridium botulinum
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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
Botulism and Bioterrorism
• 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
Mass Producing Botulinum Toxin
• Fort Detrick (1946) – bioweapon research –
1st 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
So what?
Importance of C. botulinum Research
• Bioterrorism/outbreaks
• Kerner – use in therapeutics
• Recently – BoNT as therapeutic agent for
neuromuscular disorders
Outline of Talk
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Historical background of C. botulinum
Transmission of Botulinum toxin
Molecular pathogenesis
Therapeutic uses of Botulinum toxin
Impediments in Treatment
Concluding remarks
Transmission of Botulinum Toxin
• Mostly via improperly cooked food
• Conditions to produce toxin not completely
understood
• Complex route of transmission
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Ingestion/injection
Progenitor toxin complex
Absorbed into tissue  circulated in blood
Dock onto receptors of neuron  transcytosis  binds
up acetylcholine  paralysis
Classes of Botulinum Toxin
• 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
Target Proteins of Botulinum Toxins
Serotype
Cellular Substrate
Target Cleavage Site
A
SNAP-25
Gln197-Arg198
B
VAMP/Synaptobrevin
Cellubrevin
Gln76-Phe77
Gln59-Phe60?
C1
Syntaxin 1A, 1B
SNAP-25
Lys253-Ala254
Lys252-253
D
VAMP/Synaptobrevin (18, 181)
Cellubrevin (181)
Lys59-Leu60
Ala67-Asp68
Lys42-Leu43?
E
SNAP-25(46)
Arg180-Ile181
F
VAMP/Synaptobrevin (181, 182)
Cellubrevin
Gln58-Lys59
Gln41-Lys42?
G
VAMP/Synaptobrevin
Ala81-Ala82
Figure adapted from: Aoki. 2004. Curr Med Chem. 11: 3085-3092.
Outline of Talk
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Historical background of C. botulinum
Transmission of Botulinum toxin
Molecular pathogenesis
Therapeutic uses of Botulinum toxin
Impediments in Treatment
Concluding remarks
Molecular Pathogenesis of BoNT
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
Major Steps in BoNT Action
Figure taken from: Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.
Genetic Organization of Botulinum
Locus in Clostridium botulinum
RNAP
Core
BotR/A
5’
ha operon
ntnh-bont/A operon
v
ha70
ha17
ha34
botR/A
ntnh
bont/A
Figure adapted from: Raffestin, S., et al., 2005. Molec. Microbiol. 55: 235-249.
3’
Botulinum Toxin Type A
Aoki. 2004. Curr Med Chem. 11: 3085-3092.
Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.
Figure taken from:
Arnon, et al. 2001.
Uses of Botulinum Toxin
• Bioterrorism agent – Category A
• Local paralytic agent – Botox®
• Therapeutic agent
– Neuromuscular disorders
– Pain management
BoNT as Local Paralytic Agent
• 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
BoNT/A Induces Local Paralysis
• Local effects = dose dependent
• Injection site affects physical outcome
Before Botox®
Figures adapted from: Mendez-Eastman. 2003. Plast. Surg. Nurs. 23:64-70.
After Botox®
Outline of Talk
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Historical background of C. botulinum
Transmission of Botulinum toxin
Molecular pathogenesis
Therapeutic uses of Botulinum toxin
Impediments in Treatment
Concluding remarks
BoNT as a Therapeutic Agent
• Botox® used in aesthetics
 therapeutic use in neuromuscular disorders
• BoNT/A = Botox® - Allergan, Inc.
• BoNT/B = MYOBLOC™ - Elan Pharmaceuticals
BoNT as Therapeutic Agent in
Neuromuscular Disorders
• 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
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Blepharospasm (focal dystonia)
Strabismus
Cervical dystonia
Hyperhidrosis
BoNT/A & Muscle Hyperactivity
Cervical Dystonia (CD)
• 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
Cervical Dystonia Study with
Botox® by Allergan, Inc.
• 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 6th week
Cervical Dystonia Study with
BoNT/A as Dysport®
• 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/A & Pain Management
• 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
Peripheral and Central Nervous
System Sensitization
Figure taken from: Aoki, 2003.
Botulinum Toxin A Affects
Sensitization of PNS & CNS
Figure taken from: Aoki, 2003.
Antinociceptive Activity of BoNT/A
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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.
BoNT/A Injection Reduces
Formalin-induced Pain
• 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.
BoNT/A Reduces Pain
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.
Conclusions on Therapeutics
• BoNT mechanism = specific
• Uses are diverse
– Local flaccid paralysis
– Reducing muscle spasticity
– Reducing pain
• Currently use of BoNT
– muscle disorders and associated pain
Outline of Talk
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Historical background of C. botulinum
Transmission of Botulinum toxin
Molecular pathogenesis
Therapeutic uses of Botulinum toxin
Impediments in Treatment
Concluding remarks
Impediments in Treating with BoNT
• 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
Concluding Remarks
• 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!
And remember…
Sometimes wrinkles aren’t all
that bad!
Thank you!
• 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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