1. Serotonergic Action of Tetanus Toxin.
José Aguilera and Carles Gil.
Departament de Bioquímica i Biologia Molecular Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.
Clostridial neurotoxins are powerful weapons of a limited number of bacterial species that are used against superior organisms for their expansion and reproduction. Tetanus toxin (TeTx) is constituted by two subunits: the L-chain with protease activity and responsible for the main symptoms of the tetanus disease, and the H-chain with specific functions like binding to the nervous system, axonal transport and capacity to cross trans-synaptically. Expression of the C-terminal domain (Hc) in E. coli yields a peptide with the indicated characteristics but without toxicity. In 1999, our group has first demonstrated that both TeTx and its Hc fragment inhibit serotonin (5-HT) transport in the CNS with the same effectiveness that the serotonin selective reuptake inhibitors but at greater potency, longer half life and higher specificity. We found that the action of the TeTx on the serotonergic system is based on its capacity to activate membrane receptors with tyrosine kinase activity and, by means of signal transduction mechanisms, activate phospholipase Cg, classical protein kinase C and, finally, to phosphorylate the 5-HT carrier. These results suggest that the Hc fragment could be a powerful drug able to act positively in some psychiatric pathologies. Thus, in spite of their scary history, clostridial neurotoxins are seen to positively alter neurotransmitter action. This work was supported by Grant SAF2001-2045 from the Spanish Government.

2. Management of Bladder, Prostatic and Pelvic Floor Disorders with Botulinum Neurotoxin.
A. Albanese¹ and G. Brisinda².
¹National Neurological Institute, Catholic University of Milan, Milan; ²Department of Surgery, Catholic University of Rome, Italy.
Since its introduction in the late 1970s for the treatment of strabismus and blepharospasm, botulinum toxin (BoNT) has been increasingly used in the interventional treatment of several other disorders characterized by excessive or inappropriate muscle contractions. The use of this pluripotential agent has extended to a plethora of conditions including: focal dystonia; spasticity; inappropriate contraction in most sphincters of the body such as those associated with spasmodic dysphonia, esophageal achalasia, chronic anal fissure, and vaginismus; eye movement disorders; other hyperkinetic disorders including tics and tremors; autonomic disorders such as hyperhidrosis; genitourinary disorders such as overactive and neurogenic bladder, nonbacterial prostatitis and benign prostatic hyperplasia; and aesthetically undesirable hyperfunctional facial lines. In addition, BoNT is being investigated for the control of pain, and for the management of tension or migraine headaches and myofascial pain syndrome. BoNT injections have several advantages over drugs and surgical therapies in the management of intractable or chronic disease. Systemic pharmacologic effects are rare; permanent destruction of tissue does not occur. Graded degrees of relaxation may be achieved by varying the dose injected; most adverse effects are transient. Finally, patient acceptance is high. In this paper, clinical experience over the last years with BoNT in urologically impaired patients will be illustrated. Moreover, this paper presents current data on the use of BoNT to treat pelvic floor disorders.

3. Pain Models Utilized to Assess the Mechanism of Action of Botulinum Neurotoxin Type A.
K. Roger Aoki and Joseph Francis.
Biological Sciences Dept., Allergan Inc., Irvine, CA, USA.
Current clinical experience with botulinum toxin type A (BoNT/A, BOTOX®) has demonstrated relief of pain in those conditions in which muscle contractions are not apparent, suggesting therapeutic actions of BoNT/A at nociceptive sensory nerves. Experiments were undertaken, therefore, to ascertain the mechanism by which BoNT/A could exert antinociceptive effects in preclinical models of pain. Acute and chronic pain models were selected, to assess both the selectivity and efficacy of local BoNT/A administration in alleviating the experimental pain. Acute pain was assessed with either the rodent paw withdrawal response to heat or the first phase of the formalin-induced response. Chronic pain mechanisms were evaluated in a capsaicin-induced model of secondary allodynia, in a nerve ligation (Chung) model or in a streptozotocin (STZ)-induced peripheral diabetic neuropathy model. Results from these studies demonstrate that BoNT/A can alleviate chronic pain but has no effect on acute nociception. BoNT/A appears to block peripheral sensitization by preventing the release of nociceptive neurotransmitters, through its catalytic cleavage of the target substrate, SNAP25. This direct inhibition of peripheral sensitization results in an indirect reduction of central sensitization. These preclinical results provide a hypothetical framework for understanding the clinical antinociceptive efficacy of BoNT/A.

4.Risk Factors for Botulinum Toxin Immunoresistance and Molecular Recognition of Toxin-A by Antibodies of Immunoresistant Patients.
M. Zouhair Atassi*, Behzod Z. Dolimbek*, Joseph J. Jankovic^Ç, Lance E Steward# and K. Roger Aoki#.
*Department of Biochemistry, ^Ç Parkinson's Disease Center and Movement Disorders Clinic, Department of Neurology, Baylor College of Medicine, Houston, Texas 77030; and #Allergan, Inc., Irvine, CA 92612.
We have synthesized, purified, and characterized sixty 19-residue peptides that overlapped consecutively by 5 residues and spanned the entire 848-residue heavy (H) chain of botulinum neurotoxin A (BoNT/A). We employed these synthetic peptides to map the entire H chain of BoNT/A for the regions that bind antibodies (Abs) in anti-BoNT/A antisera of humans and of different animal species and the regions recognized by T-lymphocytes in high responder mouse strains. We have also mapped the Ab-recognition profiles in sera of 28 CD patients that have become immunoresistant to BOTOX® (BoNT/A)-treatment. The pattern of recognition varied from patient to patient, but a relatively small set of peptides were recognized by most of the patients. These were peptide N25 (H chain residues 785-803), C10 (981-999), C15 (1051-1069), C20 (1121-1139) and C31 (1275-1296). We will discuss the risk factors in cervical dystonia (CD) patients that cause unresponsiveness to BOTOX® treatment as a result of immunoresistance. The results have shown that the immune response to BoNT is influenced by dose, duration of treatment, frequency of immunization, quality of the toxin, a prior immune response to an immunologically cross-reacting toxin (e.g., tetanus neurotoxin), and the MHC of the host. Once a patient becomes immunoresistant to one toxin then switching to another toxin will often be of limited and short-lived benefit, because the patient may become immunoresistant to the second toxin. This work was supported by a grant from Allergan and by the Welch Foundation due to the award to M. Z. Atassi of the Robert A. Welch Chair of Chemistry.

5. Spasticity Associated with Multiple Sclerosis.
Mike Barnes.
Hunters Moor Regional Neurological Rehabilitation Centre, Newcastle upon Tyne, UK.
In many people spasticity is the major disabling symptom in multiple sclerosis. There are very few epidemiological studies on the prevalence of troublesome spasticity in MS, but a review of the available literature indicates that, at some point in the course of the disease, around 60% of people with MS will need treatment for their spasticity. Other work has demonstrated that there is a high unmet need in this population and a considerable amount of inappropriate prescribing. Adductor spasticity is a particularly disabling problem. Muscle spasms are also a major problem for this population, often painful and often disturbing sleep. The management of spasticity remains multidisciplinary and will usually involve a range of different treatments and management strategies. Botulinum toxin has a key role to play in the overall spasticity management programme. I will summarise the available data that confirms that botulinum toxin is a useful adjunctive therapy for the management of spasticity in MS. The evidence does demonstrate that botulinum toxin has a major role to play in the management of a difficult symptom in a disabling condition.

6. Neurophysiological And Clinical Effects Of Botulinum Toxin Type A.
Alfredo Berardelli.
Department of Neurological Sciences and Neuromed Institute, University of Rome "La Sapienza".
Botulinum toxin type A acts peripherally by inhibiting acetylcoline release from the presynaptic neuromuscular terminals, thus weakening muscle contraction, and its clinical benefit depends primarily on the toxin's peripheral action. However, a number of experimental studies in animals and in human beings have provided ample evidence supporting a central action of BT-A. Botulinum toxin blocks the gamma motor endings of jaw muscles in the rat and produces parallel denervation of extrafusal and intrafusal fibers. Evidence of fusimotor denervation suggests that BT-A alters activity in muscle spindle afferents (Filippi et al 1993, Rosales et al 1996). One approach for studying muscle spindle Ia afferents in humans is to elicit the tonic vibration reflex (TVR). Trompetto et al (unpublished observations) have tested the TVR before and after BT-A injection in patients with hand dystonia. The special sensitivity of the TVR to suppression by BT-A injection -found by the authors- could be mediated by the chemodenervation of intrafusal muscle fibers, leading to a reduction in spindle inflow to the central nervous system during vibration. Studies of the reciprocal inhibition (Priori et al 1995, Modugno et al 1998) between agonist and antagonist muscles in patients with arm dystonia and in patients with essential tremor and studies of F wave in patients with focal dystonia (Wohlfarth et al 2001) suggested that BT-A injected in the upper limb muscles can modify the excitability of the spinal cord. On the contrary in patients with cranial dystonia botulinum toxin treatment has little influence upon the excitability of brainstem interneurons (Valls-Solè et al 1993, Girlanda et al 1996). Evidence of changes in cortical functional organization after BT-A has been provided by studies with the long latency reflexes (Naumann et al 1997), somatosensory evoked potentials (Kanovsky et al 1998), magnetic stimulation of cortical motor areas (Byrnes et al 1998, Gilio et al 2000) and positron emission tomography (PET) (Ceballos-Baumann et al 1997). These studies demonstrated that the injection of BT-A in the upper limbs transiently modify the topography and the excitability of the sensory-motor cortical areas. In conclusion BT-A has complex mechanisms of action. In addition to acting directly at the neuromuscular junction, the toxin alters sensory inputs to the central nervous system, thus indirectly inducing secondary central changes (Currà et al 2004). It is possible that some of the long-term clinical benefits of BT-A treatment may also reflect plastic changes in motor output after the reorganization of synaptic density.

7. Differential Movements of Synaptic Vesicles Belonging to Different Vesicle Pools.
Michael Gaffield, Silvio Rizzoli, and William J. Betz.
Department of Physiology & Biophysics, University of Colorado Medical School, Denver, Colorado, USA. Dr. Rizzoli's current address is Max-Planck-Institut fuer biophysikalische Chemie, Goettingen, Germany.
The defining feature of a chemical synapse is the collection of synaptic vesicles in the presynaptic terminal. These vesicles participate in a cycle that permits them to be used repeatedly during sustained activity. While vesicles appear to be homogeneous, both ultrastructurally and biochemically, functional studies suggest the existence of different vesicle 'pools.' For example, the 'recycling pool' is thought to comprise those vesicles that undergo exocytosis first during repetitive stimulation, before vesicles in the 'reserve pool' are mobilized. Are the pools segregated morphologically? It is natural to predict that vesicles in the recycling pool are located close to the presynaptic membrane, and reserve pool vesicles farther away. We tested this hypothesis and found that in frog motor nerve terminals recycling pool vesicles are not clustered near the sites of exocytosis, but instead are scattered, almost randomly, throughout the vesicle cluster. Next, we measured the motions of vesicles in the two pools (using the technique Fluorescence Recovery After Photobleaching (FRAP) in nerve terminals stained with a fluorescent dye, FM1-43). We found that, in resting nerve terminals, vesicles in the recycling pool are mobile, while those in the reserve pool are not. Reserve pool vesicles can be mobilized by certain drugs, like forskolin, which activate the cyclic-AMP pathway. The movements of vesicles appear to be arise from diffusion, and are not significantly perturbed by agents that interfere with the cytoskeleton. Funding for this studies was provided by the National Institutes and the Muscular Dystrophy Association.

8. Characterization of the Protein Receptor Binding Site of Botulinum Neurotoxins B and G.
T. Binz¹, A. Rummel², T. Eichner², T. Karnath¹, S. Mahrhold¹, A. Gutcaits³, T. Weil³, and H. Bigalke².
Departments of ¹Biochemistry and ²Toxicology, Hannover Medical University, Hannover; ³Merz Pharmaceuticals GmbH, Frankfurt/M, Germany.
Synaptotagmins I and II are integral synaptic vesicle proteins that have been suggested to act as protein receptors for BoNT/B and BoNT/G. The luminal segment of both isoforms that becomes accessible for BoNTs in the synaptic cleft upon neurotransmitter release has been shown to interact with the ?-trefoil (HCC)-domain of the toxins. In order to identify the synaptotagmin binding site within the HCC-domain we conducted a computer based search. Potential interaction sites were subjected to site directed mutagenesis. Various mutated BoNTs were tested for their capability to interact with synaptotagmin by GST-pull-down assays. Effects on neurotoxicity were measured at mouse hemi-diaphragm preparations. The results obtained suggest that the protein receptor interaction site lies adjacent to the established ganglioside binding pocket thus promoting rapid access to the temporarily surface exposed protein receptor. Our molecular characterization of the toxin-receptor interaction may also serve as a basis for the design of efficient binding inhibitors that simultaneously block the access to glycolipid and protein receptor. Support: German Research Council (BI 660/2-1) and Human Frontier Science Program (RGY0027/2001B).

9. Patient Selection and Functional Measures in Post-Stroke Spasticity.
Allison Brashear.
Department of Neurology, Indiana University School of Medicine, Indianapolis, IN USA.
Botulinum toxin treatment is a unique treatment for focal post-stroke spasticity which allows focal and incremental doses to the spastic limb. Patient selection and reproducible outcome measures are essential to demonstrating the clinical benefit of treatment from injections with botulinum toxin in clinical trials and in managing patients in the clinical setting. Patient selection should include those with increased tone that interferes with activities of daily living important to the patient and/or caregiver. Tone alone can not be a determinant of the need for botulinum toxin treatment. In some circumstances, increased tone may allow improved function of the spastic limb. In addition, those who expect improvement to the pre-stroke state will be disappointed. Determining which patients to treat, assessing and measuring functional outcomes will be needed to document benefit of treatment. Managing patient and caregiver expectations will also improve the benefits perceived with botulinum toxin treatments. Functional measures developed for stroke outcome studies are not sensitive nor specific enough to assess change after a focal treatment like botulinum toxin. Attention to hand hygiene, limb position, use of the limb in activities of daily living, such as dressing and eating, and assessment of pain are targets for functional improvement in patients with post-stroke spasticity of the upper limb treated with botulinum toxin. The Disability Assessment Scale (DAS) is the first measurement tool to be developed specifically for assessing change after botulinum toxin treatment in the upper limb. To date not such scale exists for the lower extremity. While the DAS is a self report tool, it is a first step in quantifying change in function after treatment. Measurement of change in function when tone is decreased with botulinum toxin treatments will provide the best indicator of success of treatment with botulinum toxin. How to quantitate improvement in a reproducible fashion in hygiene, pain, dressing and limb position and other important activities of daily living remains to be determined.

10. Structural Aspects of SNARE - Clostridial Neurotoxin Interactions.
Axel T. Brunger and Mark A. Breidenbach.
Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford, CA 94305.
Clostridal neurotoxins (CNTs) impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme-substrate interaction remote from the active site (exosites). Recently we solved the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 Å: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. We have also solved the 2.2 Å x-ray crystal structure of tetanus light chain protease (TeNT-LC). As expected, the overall structure of TeNT-LC is similar to the other known CNT light chain structures. Differences between TeNT-LC and the other CNT light chains are mainly limited to surface features such as unique electrostatic potential profiles. An analysis of surface residue conservation reveals a pattern of relatively high variability matching the path of substrate binding around BoNT/A, possibly serving to accommodate the variations in different SNARE targets of the CNT group. This work was funded by National Institute of General Medical Sciences grant 1-RO1-MH63105-01 to ATB.

11.
Ceballos-Baumann

12. Clostridial Neurotoxins: Receptors, Modes of Entry, and Detection.
Edwin R. Chapman.
Department of Physiology, University of Wisconsin, Madison, WI 53704.
Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides. Our efforts are directed toward 1) working out the precise pathways of entry for each of the toxins 2) identifying the protein components of the toxin receptor complexes 3) devising new ways to assay for toxin activity and entry into cells and 4) reconstituting toxin translocation across lipid bilayers. We will present data which address the identity of toxin receptors and the modes of entry into cells; we will also describe a FRET-based assay that can be used to monitor toxin activity and which might prove useful for high through-put screening of toxin inhibitors. These studies are supported by grants from the NIH and AHA.

13. Endocytosis and Membrane Dynamics in Motor Neurons.
Katrin Deinhardt, Stephanie Bohnert, Carole Verastegui, Otto Berninghausen(1) and Giampietro Schiavo.
Molecular Neuropathobiology Laboratory, Cancer Research UK London Research Institute, UK. (1)Department of Biological Sciences, Imperial College, London, UK.
Axonal retrograde transport is responsible for the central delivery of endogenous ligands and signalling molecules, and constitutes the gateway for the entry in the central nervous system for pathogens and virulence factors. Among these, tetanus neurotoxin (TeNT) has been recently used as a paradigm to clarify both the molecular pathogenesis of tetanus and the machinery controlling membrane dynamics and axonal transport in motor neurons (MN). TeNT binds specifically to MN presynaptic nerve terminals, where it is internalized and retrogradely transported along the axon to the cell body. Previous studies showed that the TeNT receptor complex, which is comprised of polysialogangliosides and GPI-anchored proteins, resides in lipid microdomains. We now provide experimental evidence that the internalization machinery for TeNT is clathrin-dependent. Our findings suggest that this novel internalization and sorting route is an example of lipid-raft as well as clathrin-dependent endocytosis, two pathways that have until very recently been viewed as mutually exclusive. The mixed population of TeNT transport carriers all display neutral pH, suggesting that TeNT does not enter a classical endosomal pathway that leads to degradation. However, a subpopulation of these carriers is positive for the small GTPase rab7. Rab7 is a regulator of vesicular traffic, which is involved in controlling transport to and from late endosomes. These results suggest that for TeNT trafficking, components of the classical endocytic pathway are used in a novel context. They provide insight on the coordination of this highly specialized neuronal route with other regulated endocytic processes, such as synaptic vesicle recycling, and constitutive membrane turnover in primary MNs under normal and pathological conditions.

14. Is Light Chain Subcellular Localization a Factor Influencing Botulinum Toxin Duration of Action?
Ester Fernández-Salas, Lance E. Steward, Patton E. Garay, Shiazah Malik, Ramilla O. Lewis, Joanne Wang, Helen Ho, Sarah W. Sun, Marcella A. Gilmore, Joseph V. Ordas, Joseph Francis, and K. Roger Aoki.
Biological Sciences Dept., Allergan, Irvine, CA 92612.
Botulinum neurotoxin type A (BoNT/A) is used in the treatment of neuromuscular and autonomic disorders and pain, with a therapeutic effect lasting from 3 to 12 months. Studies in animal and cell culture models suggest that this long inhibition of exocytosis is due to the persistence of the light chain (LC/A). We have reported that LC/A localizes to the plasma membrane while LC/E, with the shortest duration of effect, resides in the cytoplasm. At the plasma membrane LC/A co-localizes and interacts with SNAP25, and resides in close proximity to the SNARE complex. Cell fractionation has confirmed the presence of LC/A in the membrane fraction and the presence of LC/E in the cytoplasmic fraction. Deletions and mutations of sequences at the N- and C-terminus of LC/A produced changes in localization and activity. In conclusion, we have identified localization signals in LC/A, and are conducting studies to establish a link between LC subcellular localization and duration of effect in cells.

15. Re-Engineering The Target Specificity Of Clostridial Neurotoxins: A Route To Novel Therapeutics.
Keith A Foster¹, Frances C. G. Alexander¹, John A. Chaddock¹, Clare L. Cox¹, Caroline J. Cruttwell¹, Lyndsey Durose¹, Charlotte Heaton¹, Elizabeth Marks¹, Clifford C. Shone¹, J Mark Sutton¹, Jonathan Wayne¹, Emily J. Ford², and Duncan F. Rogers² & K. Roger Aoki³.
¹Health Protection Agency, Centre for Emergency Preparedness & Response, Porton Down, Salisbury, SP4 0JG; ²National Heart & Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY; ³Allergan Inc, 2525 Dupont Drive, Irvine, CA92612.
The ability to chemically couple proteins to LHN-fragments of clostridial neurotoxins in order to target cells other than the natural target of the neurotoxin, and thereby inhibit exocytosis, has been reported (Chaddock et al., Infect & Immun 68: 2587, 2000; Chaddock et al., Growth Factors 18: 147, 2000). This has the potential to be therapeutically beneficial where secretion plays a causative role in a disease or medical condition. Chemical coupling is, however, not a suitable basis for producing pharmaceutical agents. The production of recombinant fusion proteins containing the LHN-domain of clostridial neurotoxins and a targeting domain, and the ability of such recombinant fusion proteins to inhibit secretion from specific target cells, will be described. In particular, a novel protein consisting of the LHN-domain of botulinum neurotoxin type C and Epidermal Growth Factor (EGF) able to inhibit secretion of mucus from epithelial cells. The potential of such a molecule to prevent mucus hypersecretion in asthma and chronic obstructive pulmonary disease will be discussed. This study was supported in part by funding from Allergan Inc. and in part by the Health Protection Agency.

16. Use of BoNT for Hyperhidrosis and Gustatory Sweating.
Dee Anna Glaser.
Department of Dermatology, Saint Louis University School of Medicine, St. Louis, MO 63104
Hyperhidrosis manifests as excessive sweating, beyond the physiologic needs to maintain normal thermoregulation. Generalized sweating is usually secondary in nature whereas localized forms may be idiopathic or secondary. The differential diagnosis is extensive and requires appropriate evaluation. Approximately 2.8% of the population has hyperhidrosis (Strutton DR et al J Am Acad Dermatol(2)274-86,2004) with the most common sites being the axilla, palms, soles, face and groin. Numerous studies have demonstrated the effectiveness and safety of BoNT for the treatment of hyperhidrosis. A recent North American multi-center double-blind placebo-controlled study of 2 doses of BoNT-A in 322 patients with primary axillary hyperhidrosis confirmed the efficacy of BoNT-A with 75% of treated patients achieving a =2 point reduction in the hyperhidrosis disease severity scale. A greater than 75% reduction in sweat production was achieved in >80% of treated subjects and there was significant improvement in quality of life measurements. There were no significant differences between the 2 doses studied and the mean duration of benefit was 7 months (in press). Gustatory sweating (GS) is a common complication of surgery or injury in the region of the parotid gland, stemming from aberrant regeneration of secromotor parasympathetic neurons. GS responds very well to low doses of BoNT-A with treatment responses lasting as long as 3 years (Laccourreye et al Ann Otol Rhinol Laryngol;107:52-5,1998). BoNT-B can be used to treat hyperhidrosis but has been associated with a high incidence of side effects including dry mouth and indigestion (Nelson L et al Br J Plast Surg(58)228-32,2005, Baumann L, et al. Dermatol Surg(31)263-70, 2005.) This study was funded by Allergan, Inc.

17. Spasticity in Children with Cerebral Palsy.
H. Kerr Graham.
Hugh Williamson Gait Laboratory and Orthopaedic Department, The Royal Children's Hospital, Melbourne, Australia. Cerebral palsy encompasses a wide range of clinical phenotypes which are best classified according to the movement disorder, topographical distribution and gross motor function (GMFCS). Spasticity and spastic-dystonia are the most common movement disorders in children with cerebral palsy and contribute to a wide range of functional impairments and secondary musculoskeletal deformities. Maintenance of muscle length, avoidance of progression from dynamic to fixed deformity and maximising functional potential are recognized goals in the management of younger children with cerebral palsy. This can best be accomplished in the context of an integrated spasticity management program which includes the use of focal, regional and generalized interventions for spasticity and muscular hypertonia including such interventions as intramuscular injection of Botulinum neurotoxin A and phenol neurolysis, selective dorsal rhizotomy (SDR) and intrathecal Baclofen (ITB). At The Royal Children's Hospital, children with cerebral palsy are identified and entered in a statewide register and followed by a multidisciplinary team. All of the above spasticity management options are offered, integrated with a program of corrective orthopaedic surgery and strengthening. In this presentation, the Spasticity Compass will be described as a guide to appropriate spasticity management in children with cerebral palsy as well as the integrated management algorithm which illustrates the role of spasticity management, musculoskeletal surgery and strengthening. Funding for this study was provided by the National Research Council (NH & MRC) Clinical Centre of Research Excellence (CCRE) Grant.

18. High-Throughput Yeast-Cell Assays for BoNT Proteases.
Neil Green, Wentian Luo, and Hong Fang.
Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, Tennessee, 37232.
The seven botulinum neurotoxin light chains (BoNT/LC) (serotypes A-G) are highly specific endopeptidases, which cleave SNARE proteins that are essential for neurotransmitter release from presynaptic membranes. High-throughput cell based assays could provide a system with which to rapidly identify intracellular inhibitors of the seven LC proteases. To this end, we have developed a variety of yeast-based assays for the LCs of serotypes B, D, and G. These assays permit a genetic selection for inhibition of protease activity, and we have developed assays to genetically select for restoration of protease activity. These assays are now being used to screen for LC protease inhibitors. We have also used our assays to probe protease/substrate specificity by genetic selection inside cells. Additional yeast-based assays are under development to monitor protease activities of the remaining BoNT/LC serotypes. This study is supported by NIH Grant 5R21AI58011-02 to N.G. and NIH Grant 1R21AI062812 to H.F.

19. The Skills to Apply Botulinumtoxin - Palpation - EMG - Muscle Stimulation - Sonography.
F. Heinen, S. Berweck, A.S.Schroeder, and S.H. Lee.
Department of Pediatric Neurology and Developmental Medicine, Children's University Hospital Munich, Germany.
Targeting muscles is relevant in the treatment of children with CP (Berweck et al. Lancet Vol 363, No.9494:249-250) The variety of technical support will be demonstrated. In CP a multilevel approach is needed. This can be achieved with sonography guidance and the 'BOTOXÒ -12Plus-Concept' (more than 12 U/kg b.w. BOTOX)without affecting safety or incidence of secondary non-response due to neutralising antibodies. Patients and Methods: Analysis of 141 patients who received 349 treatments with 'new' BOTOX (83 treatments with 12 U/kg b.w. Results: Mean number of treated muscles per treatment session: 12Plus:>6 muscles. No severe side effects were observed. During a three-years-period 3 patients tested positive for secondary non-responders due to neutralising antibodies(1.3%). Conclusion: Multi-level treatment involving more muscles can be realised with sonography and the "BOTOX-12Plus-Concept". Sonography is shown as a tool for easy, quick, painless and anatomically precise, visually controlled injection of botulinum toxin. Support: Educational grants from Allergan, Elan, Ipsen, and Merz.

20. An Update on Cervical Dystonia.
Joseph Jankovic.
Department of Neurology, Baylor College of Medicine, Houston, Texas.
Chemodenervation with botulinum toxin (BTX) has become the first-line treatment for CD, resulting in safe and effective symptomatic relief of the abnormal neck movement, posture and pain [Jankovic J, J Neurol Neurosurg Psychiatry 2004;75:951-7] and in meaningful improvements in the quality of life [Jankovic J et al., Clin Neuropharmacol 2004;27:234-244]. In the longest reported follow-up study, 45 patients who received BTX treatments continuously for at least 12 years had a mean duration of response of 15.4 ± 3.4 weeks with a significant prolongation of the effect since the initial visit [Mejia NI, Vuong KD, Jankovic J, Mov Disord 2005; 20:592-7]. In a meta-analysis of 36 long-term studies involving 2,309 subjects, any mild to moderate adverse events were reported in about 25% in the BTX-A-treated group compared with 15% in the control group [Naumann M, Jankovic J, Current Medical Research and Opinion 2004;20:981-90]. Focal weakness was the only adverse event that occurred significantly more often with BTX-A treatment than control. In an observational, prospective, study of 326 patients with CD (PRO-CD) who received up to 9 treatments with BOTOX^® (mean dose/treatment 176 units) clinical responsiveness was noted in 88-100% of patients [Comella CL et al., Neurology 2004;62 (Suppl 5): A511]. The most frequently reported adverse events were dysphagia (8.5%), neck or shoulder weakness (8.4%), and neck pain (6.5%). In a double-blind, randomized, controlled trial of 80 patients with CD, Dysport, another form of BTX-A, was significantly more efficacious than placebo; 38% of Dysport group showed positive treatment response compared to 16% in the placebo group (95% C.I., 0.02-0.41) [Truong D et al., Mov Disord. 2005, in press]. Of the 1036 samples from the PRO-CD study, 1031 (99.5%) tested negative for neutralizing antibodies measured by mouse protection assay [Comella CL et al, Neurology 2004;62 (Suppl 5):A511]. In another observational study, using BTX type B (Myobloc) in 105 prospectively followed patients with CD, 24 (23%) of patients negative for BTX-B at baseline became positive at last visit (mean number of visits 5.2, range 1-12). The most frequent adverse effects attributed to BTX-B included dry mouth (14.0%) and dysphagia (12%) [Jankovic J, Hunter C, Atassi MZ, Mov Disord 2005;20(Suppl 10):S31]. There was no difference in the magnitude of improvement between the two serotypes (BTX-A, N=74; BTX-B, N=65), but adverse effects were more frequent with BTX-B (dysphagia: BTX-A = 5% vs. BTX-B = 37%, p=0.002; dry mouth: BTX-A = 5% vs. BTX-B = 43%, p< 0.01) and greater severity of constipation (p= 0.037) than those treated with BTX-A, but did not differ with respect to other tests of autonomic function [Tintner R et al., Mov Disord 2004;19(Suppl 9):S398]. Dr. Jankovic has received research grants from Allergan, Inc. and Ipsen Ltd.

21. Clinical Application of Stabilized Low Molecular Weight (150kDa) Botulinum type A Neurotoxin Preparation for Treating Muscle Hyperactivities.
Takashi Sakamoto¹, Ryuji Kaji¹, Shunji Kozaki², Keiji Oguma ³, and Tetsuhiro Harakawa⁴.
¹Tokushima University; ²Osaka Prefectural Univeristy; ³Okayama University; ⁴Chemo-Sero-Therapeutic Research Institute (Kaketsuken, Inc.), Japan.
Clinical symptoms of acute botulism by contaminated food are characterized by the early onset and the lack of immunity toward botulinum toxin even at life-threatening paralysis after exposure to a huge amount of the toxin. The clinical action of therapeutic botulinum toxin preparations is on the other hand longer than the acute botulism and may become apparent after a few weeks. The risk of developing antibodies is low, but not negligible. This discrepancy may be explained by the large molecular weight of the therapeutic preparations added by non-toxin components, which could retard the release of the 150 kDa neurotoxin or act as an adjuvant to enhance immunogenicity. The lack of non-toxin components has been believed to increase instability of the preparation. We have developed a stabilized neurotoxin with low molecular weight (150 kD) type A botulinum toxin (NTX), which can be stored at room temperature. We compared the actions of NTX and the conventional type A preparation of 900 kD (BTX) with regard to the decreasing compound muscle action potentials (CMAPs) after tibial nerve stimulation in the rat and the immunogenicity after challenging with the toxoids made from either toxin preparation of the same clinical efficacy of reducing CMAPs. We found significantly earlier onsets and increased durations of the clinical action and reduced rate of antibody development after challenging equivalent doses. After having the approval of the entire protocol by the institutional review board of Tokushima University, we then tried to use NTX for those patients with spasticity and severe dystonia refractory to BTX treatments. We observed marked clinical improvements in the walking speed, pain, and the range of motion in these patients. We believe that NTX is promising for the clinical use in large muscles with repeated doses, perhaps in spasticity. This work was supported by a Research Grant on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.

22. Update on Botulinum Toxin Treatment of Focal Dystonias (Excluding Cervical Dystonia).
Marie-Helene Marion.
Department of Neurology, St George's Hospital and Medical school, London SW17 0QT, UK.
After 15 years of development of the use of Botulinum toxin A (BTX-A) treatment in various focal dystonias, recent studies have focused on: (1) systematic reviews to determine the efficacy and safety of the treatment (Costa et al., Cochrane- Database-Syst-Rev, 2005); (2) assessment of its long term effect; (3) impact on the quality of life. and (4) determining the equivalence of dosage between the 2 formulations of the BTX-A (Botox® and Dysport®). Many neurologists are still reluctant to treat task-specific dystonias. Recent reviews on long term follow up of writer's cramps(Marion et al., Rev Neurol 159:10,923-927, 2003) and musician's cramps (Schuele et al., Neurology 64:341-343, 2005) treated with BTX should give more confidence to neurologists to use it more widely in these indications. It may be important, though, to accept that BTX treatment has its limits. The challenge in the future will be to recognise this, in order, for example, to select patients for neurosurgery such as Deep Brain Stimulation.

23. Recombinant Engineered Antibody Potently Neutralizes Known and Novel A Type Botulinum Neurotoxins. I. Geren¹, J. Lou1, C. Garcia¹, A. Razai¹, C. Forsyth¹, T. Smith², J. Brown², C. Perez², W.H.Tepp³, E.A. Johnson³, L.A. Smith², and J.D. Marks¹.
¹Dept. of Anesthesia, University of California, San Francisco, CA; ²Toxinology Division, USAMRIID, Ft. Detrick, MD; ³Dept. of Food Microbiology and Toxicology, UW Madison, WI.
To define the extent of BoNT/A diversity, more than 100 BoNT/A producing Clostridial strains were characterized by restriction mapping and DNA sequencing. Besides the known BoNT/A1 and A2 subtypes, two additional subtypes were identified, BoNT/A3 and BoNT/A4. BoNT/A2, A3, and A4 toxins differed by 10%, 15%, and 13% at the amino acid level compared to BoNT/A1. To determine the impact of subtype variability on immune recognition, we studied the binding and neutralization capacity of 8 monoclonal antibodies (mAbs) to BoNT/A1, BoNT/A2, and BoNT/A3. All 8 mAbs bound BoNT/A1 with high affinity, but 5 of the 8 mAbs showed a marked reduction in binding to BoNT/A2 and/or BoNT/A3. Binding to BoNT/A4 could not be determined due to the low amounts of toxin produced. Binding results predicted in vivo toxin neutralization; mAbs that bound A2 or A3 toxins with low affinity had minimal neutralizing capacity. To develop a recombinant BoNT/A antitoxin, molecular evolution was used to broaden mAb specificity and increase affinity, yielding a combination of three mAbs able to bind and potently neutralize BoNT/A1, A2, A3, and A4. We conclude that successful development of recombinant antitoxin requires defining subtype variability and the screening and molecular evolution of a large panel of mAbs. Supported by:DAMD17-03-C-0076, NIAID R21 AI53389-01 and UO1 AI056493.

24. Penetration and Translocation of Fluorescent Botulinum Toxins in Cultured Hippocampal Neurons.
Claudia Verderio, Giambattista Bonanno* , Cesare Montecucco§ and Michela Matteoli.
Dept. of Medical Pharmacology and CNR Institute of Neuroscience, Center of Excellence for Neurodegenerative Diseases, University of Milano; §Dip. Scienze Biomed. Sper., Univ. of Padova; *Dept. of Exp. Medicine, Center of Excellence for Biomedical Research, University of Genova.
We have shown recently that synapses of hippocampal interneurons both in culture and in situ do not express detectable levels of the SNARE protein SNAP-25. The sensitivity of excitatory and inhibitory neurons to botulinum neurotoxins type A or type E (BoNT/A and BoNT/E), which proteolyze SNAP-25, was investigated in hippocampal neurons in culture. Synaptic vesicle recycling was specifically blocked at glutamatergic but not GABAergic synapses upon 2 hour intoxication with 10-375 nM BoNT/A, whilst selectivity for glutamatergic synapses was lost after prolonged (16 hours) exposure to 100nM BoNT/A. A preferential effect at glutamatergic synapses was also observed with BoNT/E but in a narrow concentration range. The different effectiveness of BoNT/A or /E at inhibitory terminals did not result from unequal penetration or different lifetime of the toxins in interneurons, which were assessed with fluorescent BoNT derivatives. Conversely, exogenous expression of SNAP-25 within inhibitory neurons provided sensitivity to BoNT/A. Taken together, these results indicate that BoNT/A or /E inhibit preferentially the release of excitatory versus inhibitory neurotransmitters and indicate that the level of SNAP-25 expression may account for the different effects of these neurotoxins at the two types of terminals. (Supported by Telethon Italia).

25. EMG Guidance and Electrical Stimulation for Injection of Botulinum Toxin.
Nathaniel H. Mayer* and Alberto Esquenazi^.
Department of Physical Medicine and Rehabilitation, Temple University* and Moss Rehab*^, Philadelphia, Pennsylvania, 19026.
When treating muscle overactivity in the upper motoneuron syndrome (UMNS), a number of techniques for identifying injection sites for botulinum toxin have been reported. Among these techniques, electromyographic (EMG) guidance and electrical stimulation (e-stim) guidance are probably the commonest. Childers indicated that the importance of EMG or electrical stimulation guidance to treat dystonia or spastic muscles was based more on theoretical or preclinical data than on controlled clinical trials (Childers, Phys Med Rehabil Clin N Am. 14:781, 2003). Some authors have opined that needle EMG guidance is rarely required when injecting botulinum toxin (Jankovic, Muscle Nerve. 24(11):1568, 2001). Others have found relatively low rates of accuracy without EMG guidance in focal hand dystonia (Molloy et al, Neurology. 58(5): 805, 2002) or e-stim in juvenile cerebral palsy (Chin et al, J Pediatr Orthop. 25(3):286, 2005). This paper will review current evidence for methodological considerations regarding types of guidance in the clinical delivery of botulinum toxin with special emphasis on muscle identification in UMNS. Video demonstration of EMG and e-stim techniques will be incorporated into this paper for purposes of discussion. Preliminary results of a clinical study examining motor point localization versus distributed technique will also be reviewed. This study was supported by a NIDDR Model TBI Systems of Care Grant to the Moss Rehabilitation Research Institute.

26. Sprouting at the Neuromuscular Junction and Factors Influencing the Duration of Botulinum Toxin Action.
Jordi Molgó.
Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Gif sur Yvette 91198 cedex, France.
The local injection of botulinal neurotoxins (BoNTs) into adult skeletal muscles of animals and humans blocks quantal acetylcholine release, resulting in muscle paralysis. Although a marked atrophy of skeletal muscle occurs during paralysis, there is no evident damage of motor nerve terminals. The duration of neuromuscular blockade depends upon: i) BoNT serotype; ii) dose used; and iii) animal species. Higher doses are needed to paralyze amphibian, versus mammalian, skeletal muscles. According to their duration of action, BoNTs can be classified into three groups: long-lasting (BoNT/A and BoNT/C), intermediate (BoNT/B and BoNT/F), and short-lasting (BoNT/E). In mammals, neuromuscular blockade is not permanent, as functional recovery returns within a few weeks to months. It is still unclear why the recovery-time in rodents is shorter than in humans after BoNTs exposure. In mammals muscle inactivity following a single injection of BoNT/A, /C, /D or /F elicits, within 3-5 days, a nerve outgrowth or sprouting along intramuscular axons at the nodes of Ranvier (nodal sprouting), and at motor nerve terminals (terminal sprouting) of the neuromuscular junction. The Progression of events leading to the first nerve-muscle contacts, and differentiation of new endplates after BoNTs exposure is a remarkable demonstration of synaptic plasticity. Throughout this period, important communications occur between the synaptic partners involving diverse signaling molecules, and synaptic proteins. Our studies were supported in part by grants (#026065093, #0334046004) from the Direction des Systèmes de Forces et de la Prospective.

27. Characterization of Clostridial botulinum Neurotoxin Channels in Neuroblastoma Cells.
Audrey Fischer and Mauricio Montal.
Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0366.
The channel and chaperone activities of Clostridial botulinum neurotoxin (BoNT) A were investigated in neuroblastoma cells under conditions that closely emulate those prevalent at the endosome. Channel activity occurs in bursts interspersed between periods of little or no activity. The channels are voltage dependent, opening only at negative voltages to a main conductance of ~90 pS and display a conspicuous subconductance of ~10 pS. Within bursts, the channel resides preferentially in the open state. A salient feature of the BoNT channel is that it is closed at positive voltages under conditions in which the orientation and the magnitude of the pH gradient, as well as the polarity and magnitude of the membrane potential compare fairly well with those prevailing across the endosomal membrane: pH 5.3 and positive potential on the compartment containing the BoNT and pH 7.0 and negative potential on the opposite compartment. This suggests that the BoNT heavy chain channel would be closed in the endosome until it is gated by the BoNT light chain to initiate its translocation across the membrane into the cytosol. This work was supported by the U.S. Army Medical Research and Materiel Command under Contract/Grant/Intergovernement Project Order DAMD17-02-C-0106.

28. Trojan Horse or Proton Force: Finding the Right Partner(s) for Toxin Translocation.
John R. Murphy.
Section of Molecular Medicine, Department of Medicine, Boston University School of Medicine, Boston, MA 02118.
The botulinum neurotoxins, anthrax toxins, and diphtheria toxin are all known to require passage through an acidic compartment in order to deliver their respective catalytic (C) domains to the cytosol. While much is known about their mode of action and structure function relationships, little is known of the mechanism(s) by which their C-domains are translocated across a vesicular membrane and delivered to the cytosol. We have used in vitro translocation of the (C) domain of the fusion protein toxin DAB389IL-2 from purified early endosomes as a model system to examine this process. Using translocation of ADP-ribosyltransferase as an assay, a cytosolic translocation factor (CTF) complex has been purified 650-800-fold from human T-cell and yeast extracts. The heat shock protein (Hsp) 90, its yeast ortholog Hsp 82, thioredoxin reductase, and b-COP have been identified by mass spectrometry sequencing and shown to play an essential role in the delivery of the C-domain from the endosomal lumen to the external milieu. Since C-domain translocation and release from early endosomes requires a CTF-complex, we reasoned that a toxin specific motif involved in protein-protein interaction(s) was likely to be involved in the delivery process. A 10 amino acid motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema and lethal factors, and botulinum neurotoxin serotypes A, C, and D was identified by the Basic Local Alignment Search Tool, Clustal W, and Multiple Expectation maximization for Motif Elucidation computational analysis. We show that the L221E mutation in a conserved residue within this motif results in a non-toxic translocation deficient phenotype. We then constructed a gene encoding the diphtheria toxin motif which is expressed from a CMV promoter, and isolated stable transfectants of Hut102/6TG cells, Hut102/6TG-T1. In contrast to the parental cell line, Hut102/6TG-T1 cells are ca. 104 -fold more resistant to DAB389IL-2. This resistance is reversed by co-expression of siRNA directed against the motif encoding construct. We further demonstrate the specific binding of b -COP to this motif, and postulate that protein-protein interaction between this motif and b-COP is an early step in the C-domain delivery process. This study was supported by Public Health Service grant AI021628 from the National Institute of Allergy and Infectious Diseases and by NIAID Regional Center of Excellence grant AI057159.

29. Trafficking and Post-Translational Modifications of BoNT Light Chains Within Cells.
George A. Oyler¹, Yien Che Tsai², Paul S. Fishman³, Michael Adler⁴, and Randall Kincaid¹.
¹Veritas Labs, Rockville MD; ²NCI, Fredrick MD; ³Department of Neurology, University of Maryland and VAMC Baltimore; ⁴Neurotoxicology Branch, Pharmacology Division, USAMRICD, Aberdeen Proving Ground, MD.
We have investigated the cellular trafficking and post-translational modifications of botulinum neurotoxin (BoNT) light chains (Lc) for serotypes A and E. While both BoNT/A and /E Lc are endoproteases directed towards SNAP-25, they differ greatly in presynaptic terminal persistence. BoNT/A Lc is extremely stable in the presynaptic terminal while BoNT/E is short lived. To investigate trafficking of BoNT/A and /E as a mechanism of persistence, fusions to YFP have been made. Confocal microscopy reveals that both Lc A and E are membrane associated and targeted to lipid raft microdomains. Targeting to lipid rafts requires palmitoylation of cysteine residues in A and E Lc. Cysteine mutagenesis results in failure of lipid raft targeting but retained membrane association. Since the trafficking of Lc A and E is similar, other mechanisms must account for differences in persistence. Studies of ubiquitin-proteasome degradation of Lc A and E show that Lc E is much more extensively ubiquitinated than Lc A. These differences in ubiquitin proteasome degradation potentially account for the shorter half-life of Lc E. Since the extreme persistence of Lc A is a major complication of intoxication, we have developed a BoNT Lc A and E directed "designer" E3 ubiquitin ligases to facilitate the degradation of Lc A and shorten its persistence. We demonstrate that such designer E3 ligases are able to specifically ubiquitinate Lc A and E and significantly shorten the half-life of the Lc in cells. These investigations demonstrate proof of concept for novel molecular therapies of BoNT intoxication.

30. BoNT for Gastrointestinal Disorders: Therapy and Mechanisms.
P. Jay Pasricha. University of Texas Medical Branch, Galveston, TX.
Botulinum toxin (BTX) is one of the most potent inhibitors of acetylcholine from nerve endings and this accounts for its toxic properties as well as its therapeutic application in a variety of neuromuscular syndromes. This talk focuses on the growing use of BTX in the so-called "spastic" disorders of the gastrointestinal tract. These include achalasia, where the short-term efficacy of intrasphincteric injection of BTX has been well established now. However, because of the chronicity of this condition, repeated injections of the toxin may be required at regular intervals. By contrast, the relatively short duration of action may be an advantage in disorders such as chronic anal fissure, where the benefit of this therapy has now been demonstrated in hundreds of patients. There are many other sphincteric and non-sphincteric syndromes in the gut where the efficacy of this agent is being actively tested. These include non-cardiac chest pain, gastroparesis and sphincter of Oddi dysfunction. Skeletal muscle sphincters such as the upper esophageal sphincter or the external anal sphincter/ puborectalis muscle may also be targeted with good effect. In some of these conditions, local injection of BTX may serve as a useful therapeutic trial, facilitating the decision to institute more invasive forms of therapy. The cumulative short-term experience with BTX in the gut to date suggests that it is a relatively simple and safe therapy. The use of BTX represents a novel approach for gastrointestinal motility disorders and the rapidly expanding list of successful applications holds promise for more widespread use of similar agents in the future. Additional studies on long term outcome and safety are eagerly awaited. In addition to its therapeutic importance, the study of BoNT in the GI tract has interesting and important implications for understanding the full spectrum of the biological effects of this toxin. Thus, recent work from our lab demonstrates novel sites and/or mechanisms of action that have previously not been described in the skeletal muscle system. BoNT A injections result in blockade of excitatory neuromuscular transmission in the pyloric sphincter as well as a reduced response to exogenous Ach, SubP and KCl suggesting that it might directly affect smooth muscle. Further, "gene chip" studies demonstrate a striking local plasticity in the expression of several important genes that may be of importance in the regenerative response. This study was funded by grants from the U.S. Food and Drug Administration and Allergan, Inc.

31. Do Newly Formed Synaptic Contacts Account for Functional Recovery from Botulinum Toxin Type A?
Clarke R. Slater, Alexander A. Rogozhin, Ki K. Pang.
School of Neurology, Neurobiology & Psychiatry, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK .
During recovery from botulinum toxin type A (BoTxA), motor axon sprouts grow out from the neuromuscular junction (NMJ) and make new synaptic contacts with the muscle. While it is generally considered that evoked quantal release at these new sites accounts for the initial functional recovery from BoTxA, this has never been directly tested. We used focal extracellular recording of nerve-evoked endplate currents (EPCs) from mouse epitrochleoanconeus muscles to assess quantal release, from both new contacts and the original neuromuscular junction, after a single injection of BoTxA. Quantal release first appeared about 2 weeks after BoTxA at both new contacts and original NMJs and was always greater at the original NMJs. The total area of new synaptic contacts, determined on single muscle fibers from alpha-bungarotoxin labelling of AChRs, was less than 25% of that of the original NMJs. We conclude that in our material, the sprouts are not the major source of evoked quantal release during the initial recovery from BoTxA. This study was funded by The Wellcome Trust. BoTxA was kindly donated by Allergan (BOTOX®).

1. Human Antibodies that Bind Botulinum Neurotoxin A.
Sharad P. Adekar, M.D. Elias, Katherine A. Rybinski, Fetweh Al-Saleem, Andrew B. Maksymowych, Lance L. Simpson, and Scott K. Dessain.
Cardeza Foundation for Hematologic Research, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107.
Botulinum neurotoxin (BoNT) is a neuromuscular junction blocker that has been named a category A select bioterror agent. Therapeutic human antibodies can be important countermeasures to BoNT exposure. We recently published a new hybridoma method for cloning human antibodies (Dessain et al., J. Immunol. Methods (1994) vol 201, p109), which we have employed to clone human antibodies that bind BoNT serotype A (BoNT/A). Using peripheral blood lymphocytes from volunteers vaccinated with the pentavalent botulinum toxoid vaccine, we cloned two IgM antibodies that bind BoNT/A. We verified the specificity of the antibodies by serial dilution ELISA and Western blotting using whole BoNT/A and the BoNT/A C-terminal domain (HC50). We converted one of the antibodies to an IgG1 antibody that retains BoNT/A binding specificity. We are currently testing the efficacy of these antibodies in BoNT/A neutralization in vitro and in vivo. Results from these studies will be presented. This study was supported by NIH grant K08-HL04463 to S.K.D.

2. Pharmacology of BOTOX®, Dysport®, Myobloc™ and BTX-A in Animal Models of Efficacy and Safety.
K. Roger Aoki, Alan Satorius, Connie Ardila, Meenakshi Brown, Greg Nicholson and Joseph Francis.
Allergan Inc., Irvine, CA 92612-1599, USA.
Botulinum toxin type A (BoNT/A, BOTOX®) is a potent, clinically efficacious muscle relaxant. Preclinical mouse models were used to compare the characteristics of pharmacological efficacy (mouse Digit Abduction Score assay, DAS), safety (mouse intramuscular (im)-LD50) and local intermuscular diffusion (mouse DAS/muscle atrophy) of commercial formulations of BoNT/A (Dysport®, Ipsen; BTX-A, Lanzhou Institute) and BoNT/B (Myobloc™, Elan). The DAS rank order of potency (ED50, units/kg) was BOTOX® = BTX-A > Dysport® = Myobloc™. A similar result was obtained for the rank order of the calculated safety margins (SM, im-LD50/DAS ED50). Intermuscular diffusion was assessed by the threshold dose yielding ipsilateral quadriceps atrophy 2 weeks following an intra-gastrocnemius injection, and was indexed by the diffusion margin (DM, quad atrophy threshold dose/DAS ED50). The rank order for intermuscular diffusion (most ® least) was Myobloc™ > Dysport® = BTX-A > BOTOX®. These results clearly differentiate these preparations and support the concept that different products are not interchangeable.

3. Post-Licensure Experience with BabyBIG® for the Treatment of Infant Botulism: The First 18 Months.
S.S. Arnon, G. Claes, L. Juarez, J. Barash.
Infant Botulism Treatment and Prevention Program, California Department of Health Services, Richmond, CA.
On October 23, 2003 the U.S. Food and Drug Administration (FDA) approved BabyBIG® [Botulism Immune Globulin Intravenous (Human)] for the treatment of infant botulism types A and B. As of April 30, 2005 (approximately 18 months since licensure), 124 patients in the United States have been treated with BabyBIG®, 119 of whom had laboratory-confirmed type A (50 patients, 42%), type B (64 patients, 53%), or types Ba, Bf, or F (5 patients, 3%; 1 toxin type pending) infant botulism. Thus, clinical diagnostic accuracy was 96.0% (119/124). The 4 BabyBIG®-treated, but not-infant botulism patients were ultimately confirmed to have various mitochondrial disorders (n=3) and spinal muscular atrophy type 1 (Werdnig-Hoffmann disease; n=1). An additional 13 laboratory-confirmed infant botulism cases were not treated with BabyBIG® because of late notification (n=8), uncertainty as to diagnosis (n=3), or illness so mild that hospitalization did not occur (n=1). Hence, 119/133 potentially eligible cases were treated, a "case catchment" rate of 90.1%. The 119 treated patients resided in just 20 states, but more than 75% of them lived in either Northeastern or Western states. Mean length and costs of hospital stay for treated patients nationwide were 2.3 weeks and $77,800, respectively (2.3 weeks and $78,200 for type A patients; 2.3 weeks and $78,100 for type B patients). In comparison, in the pivotal clinical trial of BabyBIG® 1992-1997, the mean length and costs of hospital stay for the placebo patients were 5.7 weeks and $162,400 (2004 dollars). In California, post-licensure BabyBIG®-treated patients had a mean hospital stay of 2.2 weeks and incurred mean hospital costs of $96,500, while out-of-state BabyBIG®-treated patients had a mean hospital stay of 2.4 weeks and incurred mean hospital costs of $63,700. In comparison to the pivotal clinical trial placebo patients, thus far in the first 18 months of BabyBIG® use, the aggregate reductions achieved in length and costs of hospital stay have been 6.4 patient-years and $7,569,700 (2004 dollars). Treatment with BabyBIG® continues to reduce the length and costs of hospital stay and in its first year and a half post-licensure, reached most, but not all, laboratory-confirmed infant botulism patients in the United States. Supported by the California Department of Health Services.

4. Cross Protection of Recombinant Subunit Vaccines Against Botulism.
Michael R. Baldwin¹, William H. Tepp², Christina L. Pier², Marite Bradshaw², Mengfei Ho³, Brenda A. Wilson³, Robert B. Fritz¹, Eric A. Johnson², and Joseph T. Barbieri¹.
¹Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, W; ²Food Research Institute, University of Wisconsin at Madison, Madison, W; ³Department of Microbiology, University of Illinois at Urbana-Champaign, IL.
Conditions were established to express the receptor binding domain (HCR) of botulinum neurotoxin (BoNT) serotypes A and E in Escherichia coli. Anti-sera to HCR/A and /E neutralized BoNT lethality in mice. HCR/A cross protective properties of HCR/A and HCR/E differ. HCR/A was an effective vaccine against neurotoxin isolated from the type A-Hall strain (BoNT/A1) and the Kyoto F strain (BoNT/A2), but not against type E toxin (BoNT/E). The protection elicited by HCR/A1 to BoNT/A1 and BoNT/A2 indicates that heavy chains can elicit cross protection within sub-serotypes of BoNT. Immunization with HCR/E protected against BoNT/E or BoNT/A intoxication. The protection obtained with HCR/E is the first indication that protection can be elicited against heterologous serotypes of BoNT. These results indicate that cross protection may be less restrictive among the serotypes than previously reported and that heavy chain subunit vaccines may protect against variant serotypes and subtypes of BoNTs. This work was supported by a grant from the Great Lakes Regional Center of Excellence (GLRCE) from NIH-NIAID U54 AI057153.

5. Regulation of Chemical and Mechanical-Evoked ATP Release from Urinary Bladder Urothelium by Botulinum Toxin Type A .
Stacey Barrick¹, William de Groat², and Lori Birder^1,2.
Departments of Medicine¹ and Pharmacology², University of Pittsburgh, Pittsburgh, PA.
Intravesical injection of Botulinum neurotoxin A (BoNTA) has clinical benefit, improving bladder function in a number of bladder dysfunctions. BoNTA may have a role as an analgesic, decreasing transmitters which may contribute to sensitization of nociceptors. ATP released from urothelium is necessary for triggering reflex bladder activity and for pain behavior evoked by bladder irritation. The goal of this study was to examine the effect of BoNTA on both mechanical- and chemical-evoked ATP release from rat bladder urothelial cells. Hypotonic medium evoked release (65.1 ± 17.4 fmol ATP/100ul) which was significantly decreased with BoNTA (10nM; 2.6 ± 0.5 fmol ATP/100ul). The effect of BoNTA could also be detected with Quinacrine dihydrochloride (binds to peptide-bound ATP). Hypotonic stimuli decreased intensity of Quinacrine-staining in cultured urothelial cells. BoNTA (10nM) blocked this effect. BoNTA also decreased chemical- (capsaicin; 10?M) evoked ATP release (59.4 ± 9.8 fmol ATP/100ul; 9.3 ± 3.7 fmol ATP/100ul with (10nM) BoNTA). While a lower concentration of BoNTA (1nM) significantly decreased (69.2%) capsaicin-evoked release, hypotonic-evoked release was not affected. This demonstrates that BoNTA may have a differential effect on chemical- versus mechanical-evoked release of transmitters. The effectiveness of BoNTA to suppress urothelial-derived transmitters may extend the use to chronic pain syndromes such as interstitial cystitis. This study was supported by NIH DKR0154824.

6. Lack of Evidence of Botulinum Toxin Diffusion in Untreated Muscles in Patients with Hemifacial Spasm.
A. Berardelli, C. Lorenzano, S. Bagnato, F. Gilio.
Department of Neurological Sciences and Neuromed University of Rome "La Sapienza".
Botulinum toxin injected into a muscle may diffuse to nearby muscles thus producing unwanted effects. In patients with hemifacial spasm, we evaluated clinically and neurophysiologically, whether botulinum toxin type A (BT-A; BOTOX-ALLERGAN-) diffuses from the injection site (orbicularis oculi) to untreated muscles (orbicularis oris from the affected side and orbicularis oculi and oris from the unaffected side). We studied 36 patients with idiopathic hemifacial spasm. Botulinum toxin was injected into the affected orbicularis oculi muscle alone (at 3 standardized sites) at a clinically effective dose. Patients were studied before (T0) and 3-4 weeks after treatment (T1). We evaluated the clinical severity of spasm, the clinical effects of botulinum toxin and muscle strength in the affected and unaffected muscles. We also assessed the peak-to-peak amplitude compound muscle action potential recorded from the orbicularis oculi and orbicularis oris muscles on both sides after supramaximal electrical stimulation of the facial nerve at the stylomastoid foramen. In all patients, botulinum toxin treatment reduced muscle spasms in the injected orbicularis oculi muscle and induced no muscle weakness in the other facial muscles. The CMAP amplitude significantly decreased in the injected orbicularis oculi muscle, but remained unchanged in the other facial muscles (orbicularis oris muscle on the affected side and contra-lateral unaffected muscles). In conclusion, in patients with hemifacial spasm, botulinum toxin, at a clinically effective dose, does not induce clinical or neurophysiological signs of diffusion to the nearby untreated orbicularis oris or contra-lateral facial muscles. This study was partially sponsored by Allergan, Inc.

7. Treatment of cChronic Whiplash Associated Disorder with Botulinum toxin A - a Doubleblind Placebo Controlled Crossover Study.
Michael Binzer, Kamaran Shorsh.
Department of Neurology, Sydvestjysk Sygehus, DK-6700 Esbjerg.
21 patients with chronic whiplash associated disorder were recruited for this study . Inclusion criteria included stable symptoms for at least 6 months and previously definitively resolved litigation. Patients were randomized to treatment with either 100 units of Botulinum toxin (Botox®) or placebo in 8 standardized sites in the neck and pericranial muscles. After 5 months the treatment was reversed. Global impression, use of analgesics, visual analogue scales, range of neck movement, Beck´s depression score and the SF-36 quality of life inventory were registered at treatment start and at 1 and 5 months after both treatments. One patient dropped out of the study after the first injection for unknown reasons. 12 patients reported meaningful relief of symptoms after Botulinum toxin but not placebo, 3 patients after placebo but not Botulinum toxin, whereas 4 patients did not experience relief after any of the treatments and one patient reported relief after both treatments (c2=14.0, p=0.003). The results of the remaining variables also favored treatment with Botulinum toxin. There were few and harmless side effects with no difference between active treatment and placebo. Further studies thus seem warranted. Botox® was provided for by Allergan free of cost.

8. Modulation of Endogenous Hemichannels by Syntaxin 1A in Xenopus Oocytes.
Juan Blasi, Ashraf Muhaisen, Jordi Aleu, Adriana Raptis, Laia Bahima, Mireia Martín-Satué, Laura Texidó, Jordi Marsal and Carles Solsona.
Department of Pathology and Experiemental Therapeutics. Universyty of Barcelona. IDIBELL. c/ Feixa Llarga s/n 08907 L'Hospitalet de Llobregat. Spain.
Using two electrode voltage-clamp, we recorded endogenous hemichannel associated currents in Xenopus oocytes by lowering the extracellular calcium concentration. The activated currents were sensitive to gap junction blocking agents such as heptanol, octanol and flufenamic acid, and was blocked by pre-injecting connexin-38-antisense oligonucleotide and enhanced by the overexpression of connexin 38, indicating that this protein forming hemichannel is responsible for the low extracellular activated current in Xenopus oocytes. The amplitude of the current was enhanced in oocytes injected with the light chain of botulinium neurotoxin C1, a clostridial neurotoxin that cleaves syntaxin 1A. In syntaxin 1A-expressing oocytes, the amplitude of the current activated by low extracellular calcium concentration was reduced. The expression of either the carboxy or the amino half fragment of syntaxin 1A also reduced the hemichannel current but to a lesser extent than the full length protein. The co-expression of syntaxin 1A and Munc18a at different molar ratios revealed a modulatory effect of Munc18a on syntaxin 1A activity. Moreover, a direct correlation between the amplitude of the ion flux through hemichannels and the ratio of Munc18a:syntaxin 1A injected was observed. Taken together, these results demonstrate a modulatory role of syntaxin 1A and Munc 18a on the ionic current supported by hemichannels. Supported by grants BFI2001-3331 and BMC2002-01697 from Spanish Government.

9. Comparative Antigenicity of Three Preparations of Botulinum Neurotoxin Type A in the Rabbit.
Joerg Bluemel*, Juergen Frevert# and Anita Schwaier*.
*Merz Pharmaceuticals GmbH, Frankfurt, Germany; #Biotecon Therapeutics, Potsdam, Germany.
NT 201 is a new Botulinum Neurotoxin Type A (BoNT/A) containing preparation, free of clostridial hemagglutinins and non-toxin non-hemagglutinating proteins naturally associated with the neurotoxin. In contrast, currently marketed products containing BoNT/A generally comprise various amounts of these clostridial non-toxin proteins, which may enhance the immunogenicity during therapeutic use. To compare the immunogenicity of NT 201 with two currently marketed products, Botox® and Dysport®, the formation of specific, BoNT/A neutralizing antibodies was assessed after repeated intradermal injection in NZW rabbits. The sera were first screened with an ELISA for antibodies directed against BoNT/A. Antibody-positive sera were further tested for their potency to neutralize the paralytic activity of BoNT/A in the mouse hemidiaphragm assay. Consistent with a potential clinical relevance of the overall clostridial protein burden for the immunogenicity of BoNT/A preparations, it could be demonstrated that repeated treatment with NT 201, in contrast to Botox® or Dysport®, did not induce the formation of BoNT/A neutralizing antibodies in rabbits.

10. MALDI-TOF MS and ESI-LC-MS/MS Detection and Differentiation of the Activities of Botulinum Neurotoxins A-G.
Anne E. Boyer*, Hercules Moura*, Adrian R. Woolfitt*, Suzanne R. Kalb*, Lisa McWilliams^, Jurgen G. Schmidt†, John R. Barr*.
*Centers for Disease Control and Prevention; ^Battelle Memorial Institute; †Los Alamos National Laboratory.
Clostridium botulinum, butyricum, baratii, and argentinense, are the primary strains known to produce potent neurotoxins which cause botulism. There are seven known toxin types, A-G, of which A, B, E, and F most commonly effect humans, types C and D birds and mammals, and type G has only been implicated once in human botulism. The only accepted diagnostic assays for botulinum neurotoxin detection are the mouse bioassay and the ELISA. Both of these assays rely solely on antibodies to determine the toxin type. We have developed a method which utilizes matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) to detect the specific endoprotease activity of each toxin type (Endopep-MS). An activity based method would also favor the detection and activity typing of novel and mixed toxin types which may have escaped detection by methods that rely solely on antibodies for typing. This method is the first to use MALDI-TOF-MS to successfully detect and differentiate the endopeptidase activities of all 7 BoNT serotypes A-G. LC-ESI-MS/MS has also been optimized for the detection of BoNT-A, B, D, E, and F. The unique substrate and product masses for each serotype with MS detection, allow multiplexing, which was demonstrated by differentiating single and multiple serotypes in one reaction. The Endopep-MS assay is sensitive and specific and should facilitate diagnostics in emergency situations. Funding is through the United States government.

11. Dose consistency of botulinum toxin type A (BoNT/A; BOTOX®) for cervical dystonia.
Allison Brashear¹, Patrick Hogan², and Maureen Wooten Watts³.
¹Department of Neurology, Indiana University Medical Center, Indianapolis, IN; ²Puget Sound Movement Disorder and Headache Clinic, Tacoma, WA; ³Neurology Specialists of Dallas, Dallas, TX.
This retrospective chart review evaluated the consistency of BoNT/A (formulated as BOTOX®) doses for the treatment of cervical dystonia in clinical practice over a 2-year period. Doses and intervals between treatments were extracted from the medical records of 172 patients who had received BoNT/A at 1 of 3 sites during 1998. A total of 1059 treatments were assessed. Mean per treatment doses ranged from 241.80 U to 254.07 U. The mean interval between treatments was 108.48 days for year 1 and 114.14 days for year 2. These findings indicate that doses of, and intervals between, BoNT/A treatments for cervical dystonia were consistent over the 2-year period at these 3 institutions. The dosing consistency suggests that the majority of patients did not develop a secondary non-response to the toxin. In previous BoNT/A responders, a waning or lack of benefit may be due to neutralizing antibodies. Supported by Allergan, Inc.

12. Anti-Epileptic Effects of Botulinum Neurotoxin E.
M. Caleo¹, L. Costantin², C. Richichi3, A. Viegi¹, F. Antonucci¹, M. Funicello³, M. Gobbi³, T. Mennini³, O. Rossetto⁴, C. Montecucco⁴, L. Maffei1², A. Vezzani³, and Y. Bozzi¹.
¹Istituto di Neuroscienze C.N.R., 56100 Pisa (Italy); ²Scuola Normale Superiore, 56100 Pisa (Italy); ³Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milano (Italy); ⁴Università di Padova, 35121 Padova (Italy).
The delivery of antiepileptic agents into the seizure focus might be of potential utility for the treatment of focal onset epilepsies. Botulinum neurotoxin E (BoNT/E) causes a prolonged inhibition of neurotransmitter release following its specific cleavage of the synaptic protein SNAP-25. Here we show that BoNT/E injected into the rat hippocampus inhibits glutamate release and blocks spike activity of pyramidal neurons. The delivery of BoNT/E to the rat hippocampus dramatically reduces both focal and generalized kainic acid-induced seizures as documented by behavioral and electrographic analysis. BoNT/E treatment also prevents neuronal loss and long-term cognitive deficits associated with kainic acid seizures. We conclude that BoNT/E possesses powerful anticonvulsant actions. The financial support of Telethon (grant GGP04086) is gratefully acknowledged.

13. Case Report: Treatment of Post-Mastectomy Syndrome With Botulinum Toxin. Brad Carpentier¹ and Michael Leong².
¹Private Practice, Salinas, CA; ²Solstice Neurosciences, Inc., S.San Francisco, CA.
Myobloc® (Botulinum toxin type B or BT-B) is FDA-approved for the treatment of cervical dystonia (CD). However, Myobloc has also demonstrated utility in treating a number of chronic pain conditions, including myofascial and low back pain (Smith H et al. Clin J Pain (2002) 18(6 Suppl):S147-54), headache (Fadeyi M and Adams Q. Am J Health-Syst Pharm (2002);59:1860-1862) and neuropathic pain (Stacey B. Am J Phys Med Rehabil (2005);84:S4-S16). The following represents a case report of its use in neuropathic pain. EB is an 87 yo F with a history of breast cancer who developed right post-mastectomy syndrome resistant to oral pharmacotherapy and local anesthetic infiltrations. Examination showed sensitivity and burning in the anterior chest and radiating along the ulnar distribution of the right upper arm with associated muscle spasms along surgical scar. Initially, BT-A 100 units was injected at 4 sites in the surgical scar region. She had 2 weeks of complete relief from burning arm pain and spasm. BT-A 200 units was injected in the same fashion approximately 3 months the first injection and with 2 months of complete pain relief before a return to baseline (spasms returned at 2.5 months). The patient received Myobloc 5,000 units with similar efficacy as the prior BT-A 200 unit injection, but with longer duration in both pain relief (2.5 months) and muscle spasms (3 months). This case report demonstrates the potential use of BTs in treating neuropathic pain. Additional clinical trials are warranted. Dr. Leong was in private practice at the time of this patient's treatment.

14. A Single-Center, Dose-Comparison, Pilot Study of the Safety and Efficacy of Botulinum Toxin Type A (Bonta) in Female Subjects with Upper Facial Rhytids.
Alastair Carruthers¹ and Jean Carruthers².
¹Division of Dermatology, ²Department of Ophthalmology, University of British Columbia, Vancouver, BC, V5Z 4E1, Canada.
Purpose: To compare the safety and efficacy of 3 BoNTA regimens during simultaneous treatment of glabellar rhytids, forehead rhytids, and crow's feet. Methods: 60 patients received 16 BoNTA injections (5 in glabellar area, 6 in crow's feet, 5 in forehead) after being randomly assigned to a dose of: 2U, 4U, or 6U/injection (total dose of 32U, 64U, or 96U, respectively). Results: Interim data are available up to week 8. The incidence of improvement at week 2 was 95% with 2U and 100% with 4U or 6U. At week 8, it was 84% with 2U, 94% with 4U, and 100% with 6U. Also at week 8, the incidence of patients with = 75% improvement was significantly different between groups: 26% (2U), 61% (4U), and 71% (6U) (P=.0007). There were no serious adverse events (AEs) and no significant between-group differences in the incidence of mild and/or transient AEs. Conclusions: Interim data suggest that BoNTA has a good safety and efficacy profile for the simultaneous treatment of various upper facial rhytids at total doses of 32U, 64U, or 96U. Higher doses appear to result in greater improvements. This study was supported by Allergan, Inc.

15. Long-Term Safety Review of Subjects Treated with Botulinum Toxin Type A (Bonta) for Cosmetic Use.
Jean Carruthers¹ and Alastair Carruthers².
¹Division of Dermatology, ²Department of Ophthalmology, University of British Columbia, Vancouver, BC, V5Z 4E1, Canada.
Purpose: To assess the long-term safety of BoNTA treatment for facial rhytids. Methods: This retrospective chart review evaluated adults who had received = 10 BoNTA treatments (exclusively one formulation) from the same investigator. Results: Fifty patients treated for glabellar rhytids, crow's feet, and/or forehead rhytids were evaluated (mean age of 43 years, 92% females). They had had a median of 19 treatment sessions, with a median cumulative dose of 690U, median dose/session of 40U, and a median of 6 years (range, 3-9 years) between initial and last treatments. Overall, 99% of the 853 treatment sessions were not associated with adverse events (AEs) and 84% of patients reported no AEs. The remaining 8 patients reported a total of 10 transient AEs; 5 were considered definitely related to BoNTA (2 bilateral eyebrow ptosis, 1 eyebrow ptosis, 1 eyelid ptosis, 1 dysphagia). No AE was severe. The risk of AEs did not appear to increase with the number of treatments or be related to the interval between treatments. Conclusion: Multiple BoNTA treatments for facial rhytids have a good long-term safety and tolerability profile. This review was supported by Allergan, Inc.

16. C-Terminal Amidation of The Neuropeptide Targeting Domain of Recombinant Clostridial Endopeptidase - Ligand Fusions.
John A. Chaddock¹, Roger J. Ling¹, Clare J. Cox¹, Philip M. H. Marks¹, Elizabeth M. Marks¹, Patrick R. Stancombe¹, Jonathan M. Wayne¹, Joe Francis², Lance E. Steward², K. Roger Aoki² and Keith A. Foster¹.
¹Centre for Emergency Preparedness and Response, Health Protection Agency, Porton Down, Salisbury, SP4 0JG, UK; ²2Allergan Inc., Irvine, CA 92612-1599, USA.
Retargeting the endopeptidase domain of clostridial neurotoxins to both neuronal and non-neuronal cells is now well established (Duggan et al., J. Biol. Chem. 277:34846, 2002; Chaddock et al., Infect. Immun. 68:2587, 2000). Although, a variety of novel agents have been created by chemical conjugation techniques, full exploitation has been limited by the lack of recombinant endopeptidase-targeting ligand fusions and, in particular, the methodology needed to prepare functional fusions in which the C-terminally located targeting ligand is amidated. Since amidation is a necessary post-translational modification for many peptides, it was necessary to overcome such a methodology restriction. Here we report the successful creation, expression and purification of a fusion protein based on the LHN endopeptidase domain of serotype A and the neuropeptide substance P. Adapting amidation methodology previously described by Cottingham et al. (Nat Biotechnol. 19:974, 2001) for small peptides, a ~100 kDa, endopeptidase-active, receptor binding fusion protein has been created. This is significant step forward toward development of fully recombinant novel agents for the treatment of a range of diseases, particularly chronic pain. This study was supported by Allergan Inc.

17. The Expression and Solubility of Botulinum Neurotoxin Light Chain E.
S. Chen¹, E. A. Johnson², M. Bradshaw², C. Pier², J. T. Barbieri¹.
¹Medical College of Wisconsin, Milwaukee, W; ²Food Research Institute, University of Wisconsin, Madison, WI.
Botulinum neurotoxins (BoNT) are the most potent protein toxins for humans. There are seven serotypes of the BoNTs, A-G, based upon anti-sera neutralization. BoNT Serotype E (BoNT/E) cleaves SNAP25 between Arg180-Ile181. Sequence comparison showed that LC/E-Beluga had 92% amino acid homology with LC/E-Alaska and was nearly identical to LC/E-Iwanni. The LC/Es were expressed as His(6)-fusion proteins in E. coli, but were not soluble. Molecular mapping showed that LC/E-Beluga (1-408), which lacked a C-terminal hydrophobic region, was soluble when expressed at 18ºC, whereas LC/E-Alaska and LC/E-Iwanni were less soluble. The lowest region of amino acid homology between LC/E-Beluga and LC/E-Alaska and LC/E-Iwanni includes a short loop where 4 of 8 amino acids differ. Conversion of three amino acids of LC/E-Alaska, to the corresponding amino acids of LC/E-Beluga, increased solubility to a level approaching LC/E-Beluga(1-408). Thus, this loop and the C terminus contribute to LC/E solubility. Full-length LC/Es were purified along with LC/E(1-408) derivatives and were found to be stable at -20ºC. Each LC/E cleaved SNAP25 with similar specific activities. The double mutation, LC/E(R347A,Y350A), which is predicted to be directly involved in the catalysis, did not express detectable hydrolytic activity for SNAP25. This work was supported by a grant from the Great Lakes Regional Center for Excellence (GLRCE) from NIH-NIAID U54 AI057153.

18. Comparison of Activity of Botulinum Neurotoxin Type A Holotoxin and Light Chain Using SNAPtideTM FRET Substrates. Todd Christian, Nancy Shine, Linda Eaton, and Karen Crawford.
List Biological Laboratories, Inc., 540 Division St., Campbell, CA.
The botulinum neurotoxins are composed of two subunits, a 100 kD heavy chain and a 50 kD light chain, which are linked by a single disulfide bond. The heavy chain is responsible for binding and translocation while the light chain domain contains the enzymatic activity. The light chain is a zinc dependent metalloprotease. The eukaryotic substrate for botulinum neurotoxin type A is the synaptosomal protein SNAP-25. As previously reported, we have developed fluorescence resonance energy transfer (FRET) substrates based on SNAP-25 that are readily recognized and cleaved by the type A holotoxin and light chain. One of the substrates contains an oAbz/DNP FRET pair and the other a FITC/DABCYL FRET pair (US patent #6504006). In this study, the specific activity of the holotoxin and recombinant light chain were determined using both FRET substrates. Specific activity is expressed in terms of µmoles of SNAPtideTM cleaved per minute per milligram of enzyme. For comparison, the specific activity of holotoxin and light chain were ascertained under optimal condition for the holotoxin. The specific activity was also calculated for the light chain under its optimal conditions. A series of ZnCl2 concentrations were tested in several buffer systems to determine whether addition of ZnCl2 would improve the enzymatic activity of the light chain. In order to determine the ultimate sensitivity of this method for detecting the presence of the protease, the limit of detection (LOD) of the SNAPtide assay was established using both substrates with the holotoxin and the light chain.

19. A Review of Adult Adverse Events Associated With Botulinum Toxin Type B (Myobloc®).
Patricia Cleveland, Michael Leong, and Mike Royal.
Solstice Neurosciences, Inc., Malvern, PA.
Myobloc® (Botulinum toxin type B; Solstice Neurosciences, Inc.) is FDA-approved for the treatment of cervical dystonia (CD). We reviewed all adult adverse events (AEs) reported to the Myobloc® license holder and subsequently incorporated into periodic safety update reports submitted to the FDA since the beginning of 2001 in order to better understand the type and frequency of AE spontaneous reports. The estimated total number of patients treated during the years 2001-2004 is 94,923. The total number of cases with AEs reported (whether related or not or serious or not) was 535 (0.6%) and of these 140 (0.1%) were serious and related, inclusive of pediatric cases. Many of these cases represented initial dosing in excess of recommended starting doses. Age ranges 60³for the adult cases are as follows: 20-39 years (21%), 40-59 (49%), and (30%). Females represented approximately 70% of all serious and related AE cases with peak age representation of 40-49 years (males peak age representation was usual 60 and older). Time to first onset of AE symptoms was less than 7 days in most cases. Of the serious and related cases with listed AEs, 23/140 (16%) involved dysphagia; 15/140 (11%), dry mouth; 9/140 (6%), myasthenia; and 1/140 (1%) dyspepsia. The most common unlisted AEs (all 4 or fewer cases) included vomiting, asthenia, constipation, visual changes and dyspnea. Drs. Cleveland, Leong and Royal are employees of Solstice.

20. An Electrophysiological Study to Demonstrate In Vivo Differences Between Two Formulations of Botulinum Toxin Type A (BOTOX® and Dysport®).
Koen de Boulle², Johan Smuts¹, Riaan van Coller¹, and Patricia Barnard¹.
¹University of Pretoria, ²Dermatology Practice, Aalst,Belgium.
Currently there are two preparations of botulinum toxin type A available: BOTOX® and Dysport®. They are believed to have the same mechanism of action but there are differences in their formulation which have significance in terms of clinical effect. Defining a conversion ratio between the units of each product has long been debated. Studies addressing this issue have mostly been based on clinical outcomes which tend to have a large variability. This study uses an objective method: the amplitude of the compound muscle action potential (CMAP) of the frontalis muscle before and after injections with the two available toxins. 80 subjects were included and 160 data sets were obtained. Injections were at 3 different dosages for each (BOTOX® 10,15,20 units, Dysport® 50,75,100 units) and volumes were kept constant. Recordings were taken on day 0,7,30. The CMAP readings showed that the 2 preparations behave differently in vivo. BOTOX® had a much more pronounced effect by day 7 but by day 30 the effects were similar for both products. This study was supported by an unrestricted grant from Allergan, Inc. 

21. Quality of Life Improvements Following Botulinum Toxin A Treatment for Neurogenic Urinary Incontinence.
Pierre Denys¹, Brigitte Schurch², Emmanuel Chartier-Kastler³, Brigitte Perrouin-Verbe⁴, Stephanie Fraczek⁵, Rich Barron⁶.
¹Hôpital Raymond Poincaré, Paris, France; ²University Hospital Balgrist, Zurich, Switzerland; ³Groupe Hospitalier Pitié Salpétrière Paris, France; ⁴CHU Nantes, Nantes, France; ⁵Allergan Ltd, UK; ⁶Allergan Inc, USA.
This was a randomized, double-blind, multicenter placebo-controlled study of the effect of two different doses of botulinum toxin A (BTX-A; BOTOX®) on the Incontinence Quality of Life Instrument (I-QoL) scores of neurogenic bladder patients. Patients with urinary incontinence due to neurogenic detrusor overactivity, requiring regular clean intermittent self-catheterization and inadequately managed on oral anticholinergic therapy were randomized to receive 200U or 300U BTX-A or placebo as 30 injections of 1ml to the detrusor using cystoscopic guidance. Changes in I-QoL scores were monitored at weeks 2, 6, 12, 18 and 24 post-treatment. Enrolled were 53 spinal cord injury and 6 multiple sclerosis patients (36 male, 23 female; mean age 41.2 yrs). Statistically significant (p=0.002) mean improvements from baseline in total I-QoL scores were recorded in BTX-A treated patients, at all timepoints from weeks 2 to 24. Increases in score ranged from 18 to 28.3 (38-60% increase from baseline) and 24.6 to 32.7 (58-77% increase from baseline) in BTX-A 200U and 300U, respectively. Increases in score from baseline ranged from 17-27% in the placebo group. BTX-A therapy represents a possible therapeutic option for restoring the quality of life of patients with neurogenic urinary incontinence. This study was funded by a research grant from Allergan Ltd.

22. Muscle Fiber Orientation in Muscles Commonly Injected with Botulinum Toxin Type A.
Supreet Deshpande and Mark Gormley.
Gillette Children's Specialty Healthcare, St. Paul Minnesota, USA 55101.
Objectives: The endplate zone is assumed to be at about the midpoint of a muscle fiber. This study was designed to locate the middle of the muscle fibers of commonly injected muscles, thus identifying the endplate zone of these muscles. Design: Dissection of cadavers. Setting: Gross Anatomy Laboratory. Subjects: 4 Cadavers, 2 males and 2 females. Measurements: The proximal and distal musculotendinous junctions in muscles of the upper and lower extremities were identified. Orientation of muscle fibers was determined. Measurements using common surface landmarks were used to determine the relationship of these muscles with the landmarks (e.g. Biceps muscle bulk extends from the upper fourth to the lower fourth of the humerus). Charts were developed using these measurements so as to be able to extrapolate these measurements to other patients of varying sizes. Illustrations of muscle fiber orientation were done and the assumed location of motor endplate bands marked. Color illustrations will be shown. Conclusion: With the thought that the endplate zone is at the middle of the muscle fiber, this detailed study of muscle fibers helps identify assumed location of motor endplates of specific muscles, thereby improving technique and efficacy of Botulinum Toxin A injections.

23. Cost of Care Comparison Between Cerebral Palsy Patients Treated with Botulinum Toxin Type A (Bont/A) and Propensity Score-Matched Controls.
Michael Dickson¹, Chris Kozma², Rich Barron³, L.Andrew Koman⁴.
¹University of South Carolina, College of Pharmacy, Columbia, SC; ²Independent Outcomes Research Consultant, West Columbia, SC; ³Allergan, Inc., Irvine, CA; ⁴Wake Forest University School of Medicine, Winston-Salem, NC.
The total cost of care for patients with cerebral palsy treated with BoNT/A was compared to cerebral palsy patients not treated with BoNT/A. A retrospective pre/post design was used with nested case-control matching based on propensity scores. Cost of services, procedures performed, and diagnoses were extracted from the paid claims for South Carolina Medicaid recipients from 1995 through 2001. Conditional logistic regression was used to estimate the relationship between these variables and BoNT/A use. After 1:6 matching, 406 patients were included in the analyses (58 cases and 348 controls). Regression results indicated that only cerebral palsy diagnosis (diplegia, hemiplegia, quadriplegia), and not cost, was significantly related to BoNT/A treatment. Over the 24 months studied, BoNT/A did not add to the total cost of care. Supported by Allergan, Inc.

24. Expression and Purification of Clostridium botulinum Neurotoxin Ligand Fusions.
Lyndsey J. Durose, Caroline J. Cruttwell, Frances C.G. Alexander, Patrick R. Stancombe, Clare L. Cox, J. Mark Sutton, Jonathan M. Wayne, Clifford C. Shone, John Chaddock, and Keith A. Foster.
Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, SP4 OJG, UK.
It has been demonstrated that it is possible to retarget Clostridium botulinum neurotoxins to a variety of cell types by replacement of the Hc binding domain with alternative ligands (Duggan et al., J. Biol. Chem. 277:34846, 2002; Chaddock et al., Infect. Immun. 68:2587, 2000). To date, these studies have focused on chemical conjugation approaches to create novel agents with a predominantly neuronal target. In this study, we have engineered a codon optimised ligand-endopeptidase fusion based on the LHn fragment of serotype C for expression in Escherichia coli. The fragment includes protease sites which allow cleavage of the fusion tag and activation to the biologically active di-chain form. In this fusion, the Hc binding domain of the toxin has been replaced by a cell specific ligand, epidermal growth factor, which effectively retargets the endopeptidase activity to human epithelial cells. This approach has also been used for the generation, expression and purification of ligand fusions with other BoNT serotypes and ligands. The purification and characterisation of such ligand fusions is discussed in terms of the therapeutic potential for re-targeted toxins.

25. Effects of Intra-Articular Botulinum Toxin Type A for Sacroiliac, Cervical/Lumbar Facet and Costosternal Joint Pain and C-2 Root and Lumbar Disc Pain. Dennis D. Dykstra¹, Mark Stucky¹, Scott Schimpff¹, Maren L. Mahowald² and Jasvinder A. Singh².
¹Department of Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis, Minnesota 55455 and ²Department of Rheumatology, Veterans Administration Medical Center, Minneapolis, Minnesota 55417.
In this small case series, we evaluated the effects and safety of injecting botulinum toxin type A (BTX-A) (Botox®) into the sacroiliac joints, cervical/lumbar facets, C-2 roots, costosternal joint and lumbar disc in 9 patients with refractory pain. Preliminary data from animal studies suggest that BTX-A may provide an analgesic effect for nociceptive and neuropathic pain by inhibiting release of neuropeptides that sensitize nociceptors (Aoki, Headache. 43 (Suppl 1):S9-S15,2003). Sacroiliac joints were injected in 3 patients with 50 units of BTX-A diluted in 2cc normal saline via fluoroscopy. One cervical and 1 lumbar facet joint, 2 cervical roots, 1 costosternal joint and 1 lumbar disc were injected with 25 units of BTX-A diluted in 0.5 cc normal saline via fluoroscopy. Six of 9 patients benefited from steroid injections in the past. Data on reduction in pain severity, change in function (range of motion and improvement in activities of daily living) and adverse events were obtained. Six of 9 patients received a significant decrease in their pain (average of 3.7 points on a 0 to 10 pain scale) and improvement in function. Effects lasted on average 3 months. No side effects were seen. Based on the positive findings of this study, further randomized controlled trials for the use of BTX-A in root and joint pain are warranted.

26. Safety and Efficacy of Repeated Botulinum Toxin Type A Treatments for Focal Upper Limb Poststroke Spasticity: Results of a 12-Month Multicenter, Open-Label Trial.
Elie Elovic¹, Allison Brashear², Darryl Kaelin³, Robin McIntosh4, Jingyu Liu⁴, and Catherine Turkel4.
¹Kessler Medical Rehabilitation Research and Education Corporation, West Orange, NJ and New Jersey Medical School, Newark, NJ; ²Indiana University School of Medicine, Indianapolis, IN; ³Rehabilitation Associates of Indiana, Indianapolis, IN;. ⁴Allergan Inc., Irvine, CA.
This multicenter, open-label trial evaluated the long-term safety and efficacy of repeated doses of botulinum toxin type A (BoNTA) for the treatment of focal upper limb poststroke spasticity. All patients (n = 279) received up to 5 treatments with 200 U to 400 U BoNTA per treatment administered to the wrist, finger, and elbow flexors. Patients were retreated at physician discretion no sooner than 12 weeks after the previous treatment. In more than 13.3% of patients, no single adverse event was reported during the year-long study. Treatment-related adverse events were reported in 7% of patients, and no serious treatment-related adverse events were reported. Muscle tone in the wrist, fingers, and elbow was markedly improved from baseline at week 6 as measured by the Ashworth Scale. This improvement was sustained throughout the year-long study. Functional disability was also improved with at least 50% of patients achieving a 1-point or greater improvement at all evaluations throughout the study in their principal area of disability. Repeated treatment with BoNTA safely improves upper limb spasticity in poststroke patients. The very low incidence of treatment-related adverse events combined with a functional improvement not reported with oral spasticity agents suggests that BoNTA represents an effective and safe long-term treatment for focal poststroke spasticity in the upper extremity. This study was supported by Allergan, Inc.

27. Capture of Active Botulinum Neurotoxin (BoNT) from Infected Media using Immunosepharose Columns,
Elizabeth R. Evans, Stephen P. Kidd and Clifford C. Shone.
Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down,Salisbury, SP4 0JG, UK.
A novel capture method has been employed to retrieve active toxin from infected media using a tetravalent immunosepharose with anti- BoNT/A,B,E and F antibodies immobilised on it. This resin can be used to bind serotypes A, B, E and F, which are implicated in human disease. After capture, the bound toxin can be incubated in the presence of its substrate (SNAP-25 in the case of BoNTs/A and /E; VAMP in the case of /B and /F). The cleaved substrate can then be applied to an ELISA plate and detected using serotype-specific cleaved end antibodies. These antibodies are raised against the unique cleaved product of each individual toxin serotype. This will allow any of the 4 serotypes to be detected in a single assay format. The assay can be completed in under 6 hours, and is serotype-specific and is as sensitive as the current standard mouse bioassay. This work was funded by the Department of Health and NIAID.

28. Efficacy and Safety of Botulinum Toxin A in Reversing Urodynamic Changes Caused by Neurogenic Detrusor Overactivity.
Karel Everaerta, Brigitte Schurchb, Marianne de Sèzec, Francois Haabd, Stephanie Fraczeke.
aGent University Hospital, Gent, Belgium, bUniversity Hospital Balgrist, Zurich, Switzerland, cHôpital Pellegrin, Bordeaux, France, fHôpital Tenon, Paris, France, eAllergan Ltd, High Wycombe, UK.
This double-blind, multicenter study investigated effects of botulinum toxin A (BTX-A; BOTOX®) on bladder management in patients with neurogenic detrusor overactivity. Enrolled were 59 patients with mean detrusor overactivity duration of 63.1 months, requiring regular clean intermittent self-catheterization and inadequately managed on oral anticholinergics. Patients were randomized to receive BTX-A (200 U or 300 U) or placebo as 30 1ml injections to the detrusor. Effects on maximum cystometric capacity (MCC), reflex detrusor volume (RDV) and maximum detrusor pressure during bladder contraction (MDP) were measured at study visits for 24 weeks post-treatment. Safety assessments were conducted at each visit and presence of antibodies to BTX-A monitored at baseline and week 24. Improvements were observed in both BTX-A groups: significant increases (p=0.020) in mean MCC, with mean change from baseline ranging from 59.5-141%, and significant decreases (p=0.023) in MDP from baseline at all post-treatment timepoints. No such improvements were observed with placebo. BTX-A therapy was well tolerated, with no reported drug-related adverse events. No patient had autonomic dysreflexia or was positive for neutralizing antibodies. BTX-A may be an important therapeutic option for improving neurogenic bladder management. This study was funded by a research grant from Allergan Ltd.

29. Identification Of FGFR3 as a Putative Receptor for Botulinum Neurotoxin Type A Uptake in Neuronal Cells.
Ester Fernández-Salas, Patton E. Garay, Cory Iverson, Shiazah Z. Malik, Lance E. Steward, and K. Roger Aoki.
Biological Sciences Dept., Allergan Inc., Irvine CA 92612.
Botulinum neurotoxin type A (BoNT/A) binds to motor neurons with high affinity through interaction of the toxin binding domain with ganglioside GT1b and a protein receptor. Here we report the identification of FGFR3 as a putative BoNT/A receptor in neuronal cells. FGFR3 was identified in a complex of about 250 kDa that was immuno-reactive to BoNT/A and FGFR3 antibodies in Neuro-2A cells treated with biotinylated toxin. Competition studies showed that FGF1, FGF2, and FGF9 are able to compete toxin binding assessed by a decrease in SNAP25 cleavage. Upon BoNT/A binding, FGFR3 is phosphorylated and transduces signal as demonstrated by the upregulation of p21/waf-1. Pretreatment with a tyrosine kinase inhibitor for FGFR and PDGFR resulted in decreased toxin activity. FGFR3 is expressed in motor neurons at the neuromuscular junction and also in Neuro-2A cells that have high-affinity uptake, but is absent in cells with either low affinity uptake (SH-SY5Y) or no uptake (HIT-T15). In conclusion, we have identified FGFR3 as a putative receptor for BoNT/A. Upon toxin binding, the receptor is phosphorylated at the tyrosine kinase domain, inducing receptor-toxin internalization and subsequent traffic

30. Analgesic Properties and Mechanism of Action of Botulinum Toxin Type A (BOTOX®).
Joseph Francis, Sijun You, Alan Satorius, Connie Ardila, Meenakshi Brown, Zhiwei Li, and K. Roger Aoki.
Allergan Inc., Irvine, CA 92612-1599, USA.
Botulinum toxin type A (BoNT/A, BOTOX®) has been shown to have therapeutic benefit in the treatment of pain states (including headache). We have assessed the pharmacological efficacy and actions of single, subcutaneous (s.c.) injections of BoNT/A in rat chronic pain models. In summary, BoNT/A pretreatment dose-dependently inhibited the following: (1) capsaicin-induced thermal hyperalgesia and mechanical allodynia; (2) capsaicin-induced increased peripheral blood flow (CGRP-mediated neurogenic vasodilatation); (3) capsaicin-induced low-threshold mechanosensitive receptive field (spinal WDR neuron) expansion; and (4) streptozotocin (STZ)-induced mechanical allodynia. Single BoNT/A pretreatments with maximally efficacious doses lasted greater than 7 days. Taken together, these data further support an analgesic mechanism of action whereby BoNT/A directly inhibits peripheral nociceptor sensitization due, at least in part, to the inhibition of peripheral CGRP release, and indirectly inhibits central (spinal) sensitization. Further, these data suggest the application of BoNT/A for sustained analgesia in select chronic pain states.

31. Expression of Recombinant Di-Chain Neurotoxins. J. Frevert and V. Specht.
BioteCon Therapeutics GmbH, 14473 Potsdam, Germany.
In order to be translocated into the cytosolic compartment of motoneurons and to exhibit full proteolytic activity on their respective SNARE protein substrates, botulinum toxins must be processed to disulfide linked di-chain polypeptides. Whereas some native neurotoxins are at least partially nicked by a Clostridial protease, recombinant toxins produced in E. coli have to be nicked by trypsin or by a site-specific protease, which recognizes a cleavage site introduced by means of genetic engineering into the loop region between the light and heavy chains. We have developed neurotoxins, which are expressed by E. coli K12 derivatives as fully active, di-chain polypeptides. This could be achieved by introducing a peptide sequence into the loop region of BoNT/A that resembles the recognition sequence for thrombin. This modified loop is susceptible to a protease in the E. coli lysate, which cleaves the engineered BoNT/A in the native part of the loop sequence. Exchanging the loop regions of BoNT/B and BoNT/C1 for the modified loop of BoNT/A also made these neurotoxins a substrate for the E. coli protease. The presented method is advantageous for the production of recombinant botulinum toxins as pharmaceuticals because protease treatment for activation is avoided, facilitating purification and fulfillment of regulatory demands.

32. Botulinum Toxin Type A (BOTOX®) on Quality of Life in Chronic Whiplash.
Brian Freund and Marvin Schwartz.
The Crown Institute, Toronto, Canada.
Objective: Compare QoL impact of BOTOX® (BTX) vs. placebo in chronic whiplash. Design: Double-blind, placebo controlled, 30 subjects (12 male, 18 female), age 18-49, avg. 15 month whiplash history, initial pain of >5 based on 10-point VAS. Interventions: 10 intramuscular injections (BTX 100U or 200U, or saline-placebo) within cervical paraspinal muscle group based on tenderness to palpation; 0.2 ml/inj., 30 gauge needle. SF-36 and Pain VAS were evaluated monthly for 3 months. Results: Improvements from Baseline in SF-36 Physical Health Component at Month 3 were significantly greater in subjects receiving BTX 100U or 200U than in those receiving placebo (23.6±16.2, 23.6±15.9, 6.3±8.8, respectively; p=0.01). Improvements in SF-36 Mental Health Component with BTX 100U or 200U were significantly greater than placebo (15.1±12.5, 13.7±9.2, 2.4±6.2, respectively; p=0.009). Pain VAS decreases with BTX 100U or 200U were significantly greater than with placebo (-3.20±1.81, -2.50±0.97, -0.20±1.14, respectively; p=0.0003). Conclusion: 100U BTX provides significant improvement in QoL for patients with chronic whiplash. Supported by an unrestricted grant from Allergan, Inc.

33. Buforin Inhibitors for Botulinum Toxin B.
Gregory E. Garcia, Julie J. Ha, Deborah Moorad-Doctor, LaTawnya Y. Askins, Mark L. Jewell, Harry Singh, Ruthie H. Ratcliffe, Diane M. Calinski, John H. Carra*, and Richard K. Gordon.
Walter Reed Army Institute of Research, Division of Biochemistry, Department of Biochemical Pharmacology, Silver Spring, MD 20910-7500, * United States Army Medical Research Institute of Infectious Disease, Fort Detrick, MD 21702-5011.
Botulinum toxins (BoNT) cause flaccid paralysis by inhibiting acetylcholine release at the neuromuscular junction. The seven serotypes of BoNTs consist of a light chain (LC) containing the metalloprotease activity and a nerve-cell targeting heavy chain (HC). We have focused on BoNT/B which cleaves synaptobrevin (VAMP-2), the natural substrate of BoNT/B, between amino acids glutamine (Q) and phenylalanine (F). Buforin I (B-I), an antimicrobial peptide that contains 39 amino acids, was first isolated from the stomach of Asian toads. Since B-I contains a VAMP-2 amino acid QF sequence, we evaluated B-I as a substrate for or an inhibitor of BoNT/B. While B-I was not cleaved by BoNT/B it inhibited BoNT/B activity dose-dependently and competitively. We used the B-I analog with a CYS residue at the N-terminus as a site to fluorescently label the peptide with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Coupling efficiency was very low (~11%), perhaps due to an inability to add reducing agents as they react with the CPM sulfhydryl coupling reagent. In contrast, fluorescein-5-maleimide (F), which also couples to CYS residues, exhibited a labeling efficiency of about 80%. The resulting product, FB-I, was purified by HPLC. We analyzed the binding of FB-I using fluorescent polarization and determined that FB-I binds to the reduced holotoxin (BoNT/B, Sigma) with an EC50 of about 1 µM, similar to that obtained for native B-I inhibition of BoNT/B. To improve the potency of B-I, we constructed B-I analogs with cysteine (CYS) residues at different positions on the peptide. CYS insertions at the P-1 and P-2 sites improved potency for shorter peptide inhibitors but these were not more potent than BI. Circular dichroism (CD) spectra showed that some buforin analogs undergo conformational changes with increasing 2, 2, 2-trifluoroethanol (TFE) concentrations, which may mimic the induced fit of the inhibitors for the active site environment of the toxin, and may explain why certain buforin analogs are better inhibitors of BoNT/B. Delivery of potent BoNT inhibitors to the intoxicated neurons is a recognized problem for any intracellular therapeutic, therefore, we are determining the ability of FB-I to penetrate cultured neuroblastoma cells. FB-I was internalized by these cells as determined by confocal microscopy. Additionally, B-I was found not to be toxic at the highest concentration tested of 100 µM. Taken together, these results show that B-I exhibits two different and necessary functions for a BoNT/B therapeutic; the ability to inhibit BoNT/B, and