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  1. 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

  2. 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/

  3. And of course…Botox®Aka “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/Publications/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 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. 23:64-70.

  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 aren’t 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/Humour/MedMiscel.htm

  43. References • Aoki, K.R. 2001. A Comparison of the Safety Margins of Botulinum Neurotoxin Serotypes A, B, and F in Mice. Toxicon. 39: 1815-1820. • Aoki, K.R. 2003. Evidence for Antinociceptive Activity of Botulinum Toxin Type A in Pain Management. Headache. 43: S9-S15. • Aoki, K.R. 2004. Botulinum Toxin: A Successful Therapeutic Protein. Curr. Med. Chem. 11: 3085-3092. • Bossi, P., A. Tegnell, A. Baka, F. Van Loock, J. Hendriks, A. Werner, H. Maidhof, and G. Gouvras. 2004. Bichat Guidelines for the Clinical Management of Botulism and Bioterrorism-related Botulism. Eurosurveillance. 9: 1-4. • Breldenbach, M.A. and A.T. Brunger. 2004. Substrate Recognition Strategy for Botulinum Neurotoxin Serotype A. Nature. 432: 925-929. • Casadevall, A., E. Dadachova, and L. Pirofski. 2004. Passive Antibody Therapy for Infectious Diseases. Nat Rev Microbiol. 2: 695-703. • Casadevall, A. and L. Pirofski. 2004. The Weapon Potential of a Microbe. Trends Microbiol. 12: 259-263. • Caya, J.G., R. Agoi, and J.E. Miller. 2004. Clostridium botulinum and the Clinical Laboratorian: A Detailed Review of Botulism, Including Biological Warfare Ramifications of Botulinum Toxin. Arch Pathol Lab Med. 128: 653-662. • CDC Botulism Emergency Preparedness & Response. http://www.bt.cdc.gov/agent/botulism/

  44. References • Chaddock, J.A., J.R. Purkiss, L.M. Friis, J.D. Broadbridge, M.J. Duggan, S.J. Fooks, C.C. Shone, C.P. Quinn, and K.A. Foster. Inhibition of Vesicular Secretion In Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative of Clostridium botulinum Type A. Infect. Immun. 68: 2587-2593. • Coffield, J.A., N.M. Bakry, A.B. Maksymowych, and L.L. Simpson. 1999. Characterization of a Vertebrate Neuromuscular Junction That Demonstrates Selective Resistance to Botulinum Toxin. J. Pharmacol. Exp. Ther. 289: 1509-1516. • Coffield, J.A., N.M. Bakry, R.-d. Zhang, J. Carlson, L.G. Gomella, and L.L. Simpson. 1997. In Vitro Characterization of Botulinum Toxin Types A, C and D Action on Human Tissues: Combined Electrophysiologic, Pharmacologic and Molecular Biologic Approaches. J. Pharmacol. Exp. Ther. 280: 1489-1498. • Cui, M., S. Khanijou, J. Rubino, and K.R. Aoki. 2004. Subcutaneous Administration of Botulinum Toxin A Reduces Formalin-Induced Pain. Pain. 107: 125-133. • De Paiva, A., F.A. Meunier, J. Molgo, K.R. Aoki, and J.O. Dolly. 1999. Functional Repair of Motor Endplates After Botulinum Neurotoxin Type A Poisoning: Biphasic Switch of Synaptic Activity Between Nerve Sprouts and their Parent Terminals. Proc. Natl. Acad. Sci. USA. 96: 3200-3205.

  45. References • Erbguth, F.J. 2004. Historical Notes on Botulism, Clostridium botulinum, Botulinum Toxin, and the Idea of the Therapeutic Use of the Toxin. Mov Dis. 19: S2-S6. • Fernandez-Salas, E., H. Ho, P. Garay, L.E. Steward, and K.R. Aoki. 2004. Is the Light Chain Subcellular Localization an Important Factor in Botulinum Toxin Duration of Action? Mov. Dis. 19: S23-S34. • Fernandez-Salas, E., L.E. Steward, H. Ho, P.E. Garay, S.W. Sun, M.A. Gilmore, J.V. Ordas, J. Wang, J. Francis, and K.R. Aoki. 2004. Plasma Membrane Localization Signals in the Light Chain of Botulinum Neurotoxin. Proc. Natl. Acad. Sci. USA. 101: 3208-3213. • Foran, P.G., N. Mohammed, G.O. Lisk, S. Nagwaney, G.W. Lawrence, E. Johnson, L. Smith, K.R. Aoki, and J.O. Dolly. 2003. Evaluation of the Therapeutic Usefulness of Botulinum Neurotoxin B, C1, E, and F Compared with the Long Lasting Type A: Basis for Distinct Durations of Inhibition of Exocytosis in Central Neurons. J. Biol. Chem. 278: 1363-1371. • Franciosa, G., M. Pourshaban, A. De Luca, A. Buccino, B. Dallapiccola, and P. Aureli. 2004. Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography. App Env Microbiol. 70: 4170-4176.

  46. References • Hall, Y.H.J., J.A. Chaddock, H.J. Moulsdale, E.R. Kirby, F.C.G. Alexander, J.D. Marks, and K.A. Foster. 2004. Novel Application of an in vitro Technique to the Detection and Quantification of Botulinum Neurotoxin Antibodies. J. Immun. Met. 288: 55-60. • Hong, B., L. Jiang, Y. Hu, D. Fang, and H. Guo. 2004. J. Microbiol. Met. 58: 403-411. • Jathoul, A.P., J.L. Holley, and H.S. Garmory. 2004. Efficacy of DNA Vaccines Expressing the Type F Botulinum Toxin Hc Fragment Using Different Promoters. Vaccine. 22: 3942-3946. • Johnston, M.D., S. Lawson, and J.A. Otter. 2005. Evaluation of Hydrogen Peroxide Vapour as a Method for the Decontamination of Surfaces Contaminated with Clostridium botulinum Spores. J. Microbiol. Met. 60: 403-411. • Lacy, D.B., W. Tepp, A.C. Cohen, B.R. DasGupta, and R.C. Stevens. 1998. Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity. Nat. Struct. Biol. 5: 898-902. • Lovenklev, J. E. Holst, E. Borch, and P. Radstrom. 2004. Relative Neurotoxin Gene Expression in Clostridium botulinum Type B, Determined Using Quantitative Reverse Transcription-PCR. App Env Microbiol. 70: 2919-2927. • Maksymowych, A.B. and L.L. Simpson. 1998. Binding and Transcytosis of Botulinum Neurotoxin by Polarized Human Colon Carcinoma Cells. J. Biol. Chem. 273: 21950-21957.

  47. References • Maksymowych, A.B., M. Reinhard, C.J. Malizio, M.C. Goodnough, E.A. Johnson, and L.L. Simpson. 1999. Pure Botulinum Neurotoxin Is Absorbed from the Stomach and Small Intestine and Produces Peripheral Neuromuscular Blockade. Infect. Immun. 67: 4708-4712. • Marvaud, J.C., S. Raffestin, M. Gibert, and M.R. Popoff. 2000. Regulation of the Toxigenesis in Clostridium botulinum and Clostridium tetani. Biol. Cell. 92: 455-457. • McLaughlin, J.B., J. Sobel, T. Lynn, E. Funk, and J.P. Middaugh. 2004. Botulism Type E Outbreak Associated with Eating a Beached Whale, Alaska. Emerg. Infect. Dis. 10: 1685-1687. • Mendez-Eastman, S.K. 2003. BOTOX: A Review. Plast Surg Nurs. 23: 64-70. • Merrison, A.F.A., K.E. Chidley, J. Dunnett, and K.A. Sieradzan. 2005. Lesson of the Week: Wound Botulism Associated with Subcutaneous Drug Use. BJM. 325: 1020-1021. • Park, J. and L.L. Simpson. 2003. Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component. Infect & Immun. 71: 1147-1154. • Park, J. and L.L. Simpson. 2004. Progress Toward Development of an Inhalation Vaccine Against Botulinum Toxin. Expert Rev. Vacc. 3: 477- 487.

  48. References • Purkiss, J., M. Welch, S. Doward, and K. Foster. 2000. Capsaicin-Stimulated Release of Substance P from Cultured Dorsal Root Ganglion Neurons: Involvement of Two Distinct Mechanisms. Biochem. Pharm. 59: 1403- 1406. • Raffestin, S. B. Dupuy, J.C. Marvaud, and M.R. Popoff. 2005. BotR/A and TetR are Alternative RNA Polymerase Sigma Factors Controlling the Expression of the Neurotoxin and Associated Protein Genes in Clostridium botulinum Type A and Clostridium tetani.Molec. Microbiol. 55: 235-249. • Rohrich, R.J., J.E. Janis., S. Fagien, and J.M. Stuzin. 2003. The Cosmetic Use of Botulinum Toxin. CME Plast. Reconstr. Surg. 112: S177-S187. • Sahai, A, M. Khan, C.J. Fowler, and P. Dasgupta. 2005. Botulinum Toxin for the Treatment of Lower Urinary Tract Symptoms: A Review. Neurourology. 24: 2-12. • Scott, A.B. 2004. Development of Botulinum Toxin Therapy. Dermatol. Clin. 22: 131-133. • Simpson, L.L. 1986. A Preclinical Evaluation of Aminopyridines as Putative Therapeutic Agents in the Treatment of Botulism. Infect. Immun. 52: 858- 862. • Simpson, L.L., A.B. Maksymowych, and S. Hao. 2001. The Role of Zinc Binding in the Biological Activity of Botulinum Toxin. J. Biol. Chem. 276: 27034- 27041.

  49. References • Simpson, L.L., H. Zepeda, and I. Ohishi. 1988. Partial Characterization of the Enzymatic Activity Associated with the Binary Toxin (Type C2) Produced by Clostridium botulinum.Infect. Immun. 56: 24-27. • Simpson, L.L., J.A. Coffield, and N. Bakry. 1993. Chelation of Zinc Antagonizes the Neuromuscular Blocking Properties of the Seven Serotypes of Botulinum Neurotoxin as well as Tetanus Toxin. J. Pharmacol. Exp. Ther. 267: 720-727. • Simpson, L.L., Y. Kamata, and S. Kozaki. 1990. Use of Monoclonal Antibodies as Probes for the Structure and Biological Activity of Botulinum Neurotoxin. J. Pharmacol Exp Ther. 255: 227-232. • Sobel, J., N. Tucker, A. Sulka, J. McLaughlin, and S. Maslanka. 2004. Foodborne Botulism in the United States, 1990-2000. Emerg. Infect. Dis. 10: 1606-1611. • Ting, P.T. and A. Freiman. 2004. The Story of Clostridium botulinum: from Food Poisoning to Botox. Clin. Med. 4: 258- 261. • Truong, D., D.D. Duane, J. Jankovic, C. Singer, L.C. Seeberger, C.L. Comella, M.F. Lew, R.L. Rodnitzky, F.O. Danisi, J.P. Sutton, P.D. Charles, R.A. Hauser, G.L. Sheean. Efficacy and Safety of Botulinum Type A Toxin (Dysport) in Cervical Dystonia: Results of the First US Randomized, Double-blind, Placebo-controlled Study. 2005. Mov. Dis.

  50. References • Yoneda, S., M. Shimazawa, M. Kato, A. Nonoyama, Y. Torii, H. Nishino, N. Sugimoto, S. Kozaki, and H. Hara. 2004. Comparison of the Therapeutic Indexes of Different Molecular Forms of Botulinum Toxin Type A. Eur. J Pharm. 508: 223- 229. • Yowler, B.C. and C. Schengrund. 2004. Glycosphingolipids—Sweets for Botulinum Neurotoxin. Glycoconjugate. 21: 287-293. • Zhang, L., W. Lin, S. Li, and K.R. Aoki. 2003. Complete DNA Sequences of the Botulinum Neurotoxin Complex of Clostridium botulinum Type A-Hall (Allergan) Strain. Gene. 315: 21-32.