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Title: Clostridium botulinum Toxin: The Neuromuscular Wonder Drug


1
Clostridium botulinum ToxinThe Neuromuscular
Wonder Drug
  • Amy Malhowski
  • Biology 360
  • March 30, 2005

Figure taken from http//www.consultingroom.com/A
esthetics/Products/Product_Display.asp?ID1
2
Public Perception of Botulinum Toxin
Bioterrorism!
Figures taken from http//www.safetycentral.com/b
ottoxfacin.html http//archives.cnn.com/2002/ALLPO
LITICS/06/12/bush.terror/
3
And of courseBotoxAka The Fountain of Youth
Figure taken from http//lingua.utdallas.edu7000
/1442/
4
Outline of Talk
  • Historical background of C. botulinum
  • Transmission of Botulinum toxin
  • Molecular pathogenesis
  • Therapeutic uses of Botulinum toxin
  • Concluding remarks

5
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

6
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.
7
Symptoms of Botulism
Figure taken from Caya, et al., 2004.
8
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

9
Clostridium botulinum
  • 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/Publication
s/Special/cover2.htm
10
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

11
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

12
So what?Importance of C. botulinum Research
  • Bioterrorism/outbreaks
  • Kerner use in therapeutics
  • Recently BoNT as therapeutic agent for
    neuromuscular disorders

13
Outline of Talk
  • Historical background of C. botulinum
  • Transmission of Botulinum toxin
  • Molecular pathogenesis
  • Therapeutic uses of Botulinum toxin
  • Impediments in Treatment
  • Concluding remarks

14
Transmission of Botulinum Toxin
  • 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

15
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

16
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.
17
Outline of Talk
  • Historical background of C. botulinum
  • Transmission of Botulinum toxin
  • Molecular pathogenesis
  • Therapeutic uses of Botulinum toxin
  • Impediments in Treatment
  • Concluding remarks

18
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

19
Major Steps in BoNT Action
Figure taken from Simpson. 2004. Annu. Rev.
Pharmacol. Toxicol. 44 161-193.
20
Genetic Organization of Botulinum Locus in
Clostridium botulinum
v
RNAP Core
BotR/A
5
3
ha operon
ntnh-bont/A operon
botR/A
ntnh
bont/A
ha34
ha17
ha70
Figure adapted from Raffestin, S., et al., 2005.
Molec. Microbiol. 55 235-249.
21
Botulinum Toxin Type A
Aoki. 2004. Curr Med Chem. 11 3085-3092.
Simpson. 2004. Annu. Rev. Pharmacol. Toxicol.
44 161-193.
22
Figure taken from Arnon, et al. 2001.
23
Uses of Botulinum Toxin
  • Bioterrorism agent Category A
  • Local paralytic agent Botox
  • Therapeutic agent
  • Neuromuscular disorders
  • Pain management

24
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

25
BoNT/A Induces Local Paralysis
  • Local effects dose dependent
  • Injection site affects physical outcome

After Botox
Before Botox
Figures adapted from Mendez-Eastman. 2003.
Plast. Surg. Nurs. 2364-70.
26
Outline of Talk
  • Historical background of C. botulinum
  • Transmission of Botulinum toxin
  • Molecular pathogenesis
  • Therapeutic uses of Botulinum toxin
  • Impediments in Treatment
  • Concluding remarks

27
BoNT as a Therapeutic Agent
  • Botox used in aesthetics
  • ? therapeutic use in neuromuscular disorders
  • BoNT/A Botox - Allergan, Inc.
  • BoNT/B MYOBLOC - Elan Pharmaceuticals

28
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
  • Blepharospasm (focal dystonia)
  • Strabismus
  • Cervical dystonia
  • Hyperhidrosis

29
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

30
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

31
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

32
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

33
Peripheral and Central Nervous System
Sensitization
Figure taken from Aoki, 2003.
34
Botulinum Toxin A Affects Sensitization of PNS
CNS
Figure taken from Aoki, 2003.
35
Antinociceptive Activity of BoNT/A
  • 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.
36
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.
37
BoNT/A Reduces Pain
  • 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.
38
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

39
Outline of Talk
  • Historical background of C. botulinum
  • Transmission of Botulinum toxin
  • Molecular pathogenesis
  • Therapeutic uses of Botulinum toxin
  • Impediments in Treatment
  • Concluding remarks

40
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

41
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!

42
And remember
Sometimes wrinkles arent all that bad!
43
Thank you!
  • Chris White-Ziegler
  • My readers Caitlin Reed Natalia Grob
  • Bio 360 students

Figure taken from http//www.jwolfe.clara.net/Hum
our/MedMiscel.htm
44
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