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
Figures taken from
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?

• Results in flaccid paralysis of muscles
• Caused by toxin produced from C. botulinum
• Three types via route of entry of bacteria
• Foodborne, infant, wound
• Mainly exists as foodborne outbreaks
• Now have bioterrorism threat

6
The History of Botulinum Toxin

• Coined botulism in 1700s from botulus (sausage)
  after an outbreak from consumption of 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
• 1st to connect botulism to bacterium isolated
  from raw, salted pork postmortem tissues of
  botulism victims
• Sucessfully 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

• Botulinum toxin attempted use as biological
  weapon during WWII aborted when toxin did not
  affect test animals (donkeys)
• Great potential in toxicity
• BoNT no longer considered good biological weapon

11
Mass Producing Botulinum Toxin

• Fort Detrick (1946) bioweapon research
• 1st time mass produce toxin
• Production process- grow, crystallize
• 1972 Nixon terminates all research on
  biological warfare agents
• Research continues 1979 Schantz produces
  batch 79-11 used until 1997
• 1991 several batches made Botox by Allergan
  Inc.

12
So what?Importance of C. botulinum Research

• Bioterrorism/outbreaks
• Kerner brought about idea of therapeutics
• Most recent work harnessing 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

• Most commonly via improperly cooked food
• Conditions to produce toxin not completely
  understood
• Complex route of transmission
• Ingestion/injection
• Neurotoxin produced as progenitor complex
• Absorbed into tissue ? circulates blood
• Docks 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
Botulinum Toxin Type A
Aoki. 2004. Curr Med Chem. 11 3085-3092.
Simpson. 2004. Annu. Rev. Pharmacol. Toxicol.
44 161-193.
21
Figure taken from Arnon, et al. 2001.
22
Uses of Botulinum Toxin

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

23
BoNT as Local Paralytic Agent

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

24
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.
25
Outline of Talk

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

26
BoNT as a Therapeutic Agent

• Botox used for aesthetics ? therapeutic use in
  neuromuscular disorders
• BoNT/A Botox from Allergan, Inc.
• BoNT/B MYOBLOC from Elan Pharmaceuticals

27
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 (aka focal dystonia)
• Strabismus
• Cervical dystonia
• Hyperhidrosis

28
BoNT/A Muscle Hyperactivity Cervical Dystonia
(CD)

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

29
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 suggested that majority of patients had
  beneficial response by 6th week

30
Cervical Dystonia Study with BoNT/A as Dysport

• A 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 was 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

31
BoNT/A Pain Management

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

32
Peripheral and Central Nervous System
Sensitization
Figure taken from Aoki, 2003.
33
Botulinum Toxin A Affects Sensitization of PNS
CNS
Figure taken from Aoki, 2003.
34
Antinociceptive Activity of BoNT/A

• Acute pain (phase 1) is not relieved by BoNT/A
• Inflammatory pain (phase II) is relieved by
  BoNT/A
• Increasing doses decrease phase II pain
  appreciably
• Antinociceptive activity is maintained longer
  with higher dose of BoNT/A

Figure taken from Aoki, 2003.
35
BoNT/A Injection Reduces Formalin-induced Pain

• Upon formalin challenge 5 days post-injection,
  dose-dependent decrease in Glut release is
  observed
• Injection of BoNT/A prevents increase of
  formalin-induced Glut release

Figure taken from Aoki, 2003.
36
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.
37
Conclusions on Therapeutics

• BoNT mechanism specific
• Uses are diverse
• Local flaccid paralysis
• Reducing muscle spasticity
• Reducing pain
• Currently, BoNT therapy on muscle disorders and
  associated pain

38
Outline of Talk

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

39
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
• not up to snuff on research
• stigma in using deadly toxin for good use

40
Concluding Remarks

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

41
And remember
Sometimes wrinkles arent all that bad!
42
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
43
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44
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45
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51
Miscellaneous Info.
52
BoNT in Treating Lower UTIs

• Describe disease
• What toxin does as treatment
• After effects of toxin how change disorder
• Show mechanism and data to support effects

53
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.
54
Cervical Dystonia Study with Botox
Placebo N82 Botox N88 95 CI on Difference
Baseline CDSS 9.3 9.2
Change in CDSS at week 6 -0.3 -1.3 (-2.3, 0.3) a,b
Patients with Improvement on PGAS 31 51 (5, 34) a
Pain Intensity Baseline 1.8 1.8
Change in Pain Intensity at week 6 -0.1 -0.4 (-0.7, -0.2) c
Pain Frequency Baseline 1.9 1.8
Change in Pain Frequency at week 6 -0.0 -0.3 (-0.5, -0.0) c
Table adapted from BOTOX insert, Allergan, Inc.
1998.
55
Transcytosis of BoNT

• BoNT targets gut epithelial
• Absorptive enterocytes
• M cells of Peyers Patches

Figures taken from Simpson. 2004. Annu. Rev.
Pharmacol. Toxicol. 44 161-193.
56
Major Steps in BoNT Action
Binding
Productive Internalization
Intracellular Poisoning
Low/High Affinity Site
Endocytosis
Transcytosis
Enzymatic Activity (SNAP-25, VAMP Syntaxin)
Binding (Cleavage Site)
Renature Light Chain
Expose Occult Domains
Insert Into Membrane
Reduce Disulfide Bond
Cross Membrane
Chain Separation
Dissociation of Zinc
Reassociation of Zinc
Figure adapted from Simpson. 2004. Annu. Rev.
Pharmacol. Toxicol. 44 161-193.