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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45
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