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WO2002089834A1 - Bont/e ou snap-25e utilises pour traiter une intoxication a la toxine botulique a ou c1 et inhiber la contraction musculaire - Google Patents

Bont/e ou snap-25e utilises pour traiter une intoxication a la toxine botulique a ou c1 et inhiber la contraction musculaire Download PDF

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WO2002089834A1
WO2002089834A1 PCT/GB2002/002087 GB0202087W WO02089834A1 WO 2002089834 A1 WO2002089834 A1 WO 2002089834A1 GB 0202087 W GB0202087 W GB 0202087W WO 02089834 A1 WO02089834 A1 WO 02089834A1
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bont
snap
snare
cell
patient
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Godfrey Lisk
Patrick Foran
Frederic Andre Meunier
James Oliver Dolly
Gregory O'sullivan
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Ip2ipo Innovations Ltd
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Imperial College Innovations Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to botulinum toxins (BoNTs) and their use in medicine.
  • Botulism is a paralyzing disease caused by the toxin of Clostridium botulinum (see Chermgton (1998) Muscle Nerve 21(6), 701-710 for a review).
  • the toxin produces skeletal muscle paralysis by producing a presynaptic blockade to the release of acetylcholine.
  • the several types of botulinum neurotoxin act at the nerve terminal. Since the discovery of the toxin about 100 years ago, five clinical forms of botulism have been described: 1) classic or foodborne botulism; 2) wound botulism; 3) infant botulism; 4) hidden botulism; 5) inadvertent botulism. A clinical pattem of descending weakness is characteristic of all five forms.
  • BoNTs Seven homologous serotypes of BoNT, termed A-G, are produced by different Clostridium botulinum; each has a molecular weight of about 150 kD and consist of a heavy and light chain (LC) linked by a disulphide bridge and non-covalent bonds.
  • BoNTs target motor nerve endings by binding avidly to distinct ecto-acceptors, exclusively located on cholinergic presynaptic membranes, with subsequent acceptor-mediated uptake and translocation to the cytosol where they block transmitter release (Dolly et al (1994)).
  • Both A and E cleave SNAP-25, within the C-terminus at peptide bonds (Glnl97_Argl98 mc ⁇ A r gl80_n e 181 3 respectively) in close proximity to each other, but strikingly induce neuromuscular paralysis of long (several weeks) and short (few days) durations (Eleopra et al, 1998; see also Keller et al (1999) FEBS Lett 456, 137-142).
  • SNAP-25 syntaxinl and synaptobrevin are termed SNAREs (soluble NSF- attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein).
  • SNAP-25 and syntaxinl are target membrane SNAREs (tSNAREs) whereas synaptobrevin is a vesicle-membrane SNARE (vSNARE).
  • tSNAREs target membrane SNAREs
  • vSNARE vesicle-membrane SNARE
  • Multiple isoforms of vSNARES and tSNARES have been described (which are reviewed briefly in, for example, Gonelle-Gispert et al (1999) Biochem J 339, 159-165 and more extensively in Linial (1997) J Neurochem 69, 1781-1792).
  • WO01/18038 describes methods for inhibiting SNARE-dependent exocytosis in a cell wherein a fragment, variant, chimaera (fusion; tagged) or derivative of a SNARE or a chimaera (fusion) of such a fragment, variant or derivative (inhibitory SNARE) that is capable of inhibiting SNARE- dependent exocytosis is supplied to the cell, for example in a patient.
  • This method of inhibiting exocytosis may be useful as an alternative to the inhibition of exocytosis by the administration of a clostridial toxin to cells susceptible to such inhibition by a clostridial toxin.
  • it may be useful in inhibiting exocytosis in cells that are not susceptible to clostridial toxin in vivo. Methods of reversing such inhibition of exocytosis may be useful.
  • W095/17904 suggests the use of BoNT/F when a short duration of action is required.
  • W09 4/28923 suggests the use of combinations of botulinum toxins in order to control the duration of therapeutic activity. There is no suggestion that the combination may have a shorter duration of therapeutic activity than that of the component toxins when administered individually.
  • this treatment When treating botulism, this treatment may afford relatively fast rescue of transmitter release, alleviating the symptoms when most severe and taking the patient out of the critical state. Furthermore, this treatment may preempt BoNT/A-induced nerve sprouting and long-term remodelling of the motor endplates (de Paiva et al (1999) Proc. Natl Acad. Sci. (USA) 96, 3200-3205) and may avoid the poisoning-associated extensive atrophy of the muscle fibres and negate the need for months of rehabilitation.
  • the methods may be useful in reversing therapeutic inhibition of exocytosis, for example when the inhibition is more severe than required, or has been generated in the wrong cells, for example in the wrong muscle group.
  • BoNT/E botulinum toxins
  • a first aspect of the invention provides a method for treating a patient with Botulinum toxin A (BoNT/A) or Botulinum toxin Cl (BoNT/Cl) poisoning, wherein the patient is administered Botulinum toxin E (BoNT/E).
  • Botulinum toxin A BoNT/A
  • Botulinum toxin Cl BoNT/Cl
  • BoNT/E Botulinum toxin E
  • a second aspect of the invention provides the use of BoNT/E in the manufacture of a medicament for the treatment of a patient with BoNT/A or BoNT/Cl poisoning.
  • a third aspect of the invention provides a method for treating a patient in need of reversal of inhibition of exocytosis in a cell of the patient caused by contact of BoNT/A or BoNT/Cl with the said cell, wherein the patient is administered BoNT/E.
  • a fourth aspect of the invention provides the use of BoNT/E in the manufacture of a medicament for the treatment of a patient in need of reversal of inhibition of exocytosis in a cell of the patient caused by contact of BoNT/A or BoNT/Cl with the said cell.
  • BoNT/A poisoning is more prevalent than BoNT/Cl poisoning; it is therefore preferred that the toxin is BoNT/A.
  • the BoNT/E is supplied to affected cells of the patient, as discussed further below.
  • Treatment with BoNT/E may be useful in treatment of BoNT/A or BoNT/Cl poisoning because it may prevent or diminish nerve cell sprouting (as described in Example 1) and the resultant, highly undesirable need for months of rehabilitation. In the absence of significant nerve cell sprouting, recovery of muscle function may to be quicker and more complete than when significant sprouting has taken place.
  • the active ingredient may alternatively be a polynucleotide encoding and capable of expressing BoNT/E, or SNAP-25j ⁇ or a polynucleotide encoding and capable of expressing SNAP-25 ⁇ (as discussed further below).
  • a fifth aspect of the invention provides the use of an agent which is capable of (1) reducing the amount of a fragment, variant, chimaera or derivative of a SNARE or a chimaera of a said fragment, variant or derivative (inhibitory SNARE) that is capable of inhibiting SNARE-dependent exocytosis in a cell in which an inhibitory SNARE is present, and/or (2) altering the location of the inhibitory SNARE in a cell in which an inhibitory SNARE is present, in the manufacture of a medicament for the treatment of a patient in need of reversal of inhibition of SNARE-dependent exocytosis in a cell in which the inhibitory SNARE is present.
  • a sixth aspect of the invention provides a method for reversing the inhibition of SNARE (soluble (N-ethylmaleimide-sensitive fusion protein- attachment protein receptor)- dependent exocytosis in a cell in which a fragment, variant, chimaera or derivative of a SNARE or a chimaera of a said fragment, variant or derivative (inhibitory SNARE) that is capable of inhibiting SNARE-dependent exocytosis is present, the method comprising the step of supplying to the cell an agent which is capable of reducing the amount of the inhibitory SNARE in the cell and/or altering the location of the inhibitory SNARE in the cell, wherein the method is performed in vivo, or alternatively wherein the inhibitory SNARE is present in the cell as a result of circumstances other than exposure of the cell to BoNT/A (or preferably any clostridial, for example botulinum, toxin, for example BoNT/Cl). In the latter case the method may be performed in vivo
  • SNARE soluble (N-ethylmaleimide-sensitive fusion protein- attachment protein receptor) is well known to those skilled in the art, for example Gonelle-Gispert et al (1999) Biochem J 339, 159-165 and Linial (1997) JNeurochem 69, 1781-1792.
  • SNARE polypeptides are considered to be involved in Ca2+-_egulated exocytosis, for example release of neurotransmitters from nerve terminals, insulin (stored in large dense-core granules) release, for example from pancreatic B cells or the HIT (hamster) or RIN (rat) insulin-secreting cell lines and evoked exocytosis from chromaffin cells.
  • Chromaffin cells are the secretory cells of the adrenal medulla. It is preferred that the said cell is one in which it is desirable to reduce inhibition of Ca2+-regulated exocytosis arising from the presence of the inhibitory SNARE in the cell.
  • the cell may be a cell in which the inhibitory SNARE is present as a result of exposure of the cell to a clostridial toxin, for example a botulinum toxin, for example BoNT/A or BoNT/Cl.
  • the cell may be a cell in which a clostridial toxin is capable of inhibiting Ca2+-regulated exocytosis.
  • the cell may be a cholinergic neuron.
  • the clostridial toxin is a tetanus toxin
  • the cell may be an inhibitory neuron in the spinal cord.
  • the inhibitory SNARE is present in the cell as a result of circumstances other than exposure of the cell to a clostridial toxin, for example BoNT/A or BoNT/Cl.
  • the inhibitory SNARE may be present in the cell as a result of supply of the inhibitory SNARE to the cell, for example by expression of the inhibitory SNARE in the cell from a recombinant polynucleotide or as a result of administration of the inhibitory SNARE to the cell, for example as described in WO01/18038.
  • the said cell (for example, cell in a patient) is of a type that is capable of performing SNARE-dependent exocytosis in the absence of the inhibitory SNARE. It may be a nerve cell (for example a cholinergic nerve cell or an inhibitory interneurone), adreno-chromaffin cell, insulin-secreting cell (for example a pancreatic B cell), endocrine cell lines of intestinal origin (for example cholecystokinin (CCK)-secreting cells, similar to cell lines STC-1 and GLUTag; see, for example Nemoz-Gaillard et al (1998) FEBS Lett 425(1), 66-70) or endocrine non-intestinal cell lines (similar to, for example cell lines CA-77 and HIT-T15).
  • a nerve cell for example a cholinergic nerve cell or an inhibitory interneurone
  • adreno-chromaffin cell for example a pancreatic B cell
  • insulin-secreting cell for example
  • inhibition of exocytosis in a cholinergic nerve cell by supply of an inhibitory SNARE may be useful in producing paralysis, for example localised paralysis, in a manner similar to the use of BoNT/A for the treatment of muscular movement disorders or for cosmetic treatment, for example in which facial muscles are relaxed.
  • This may be useful in, for example, patients that cannot be successfully treated using a clostridial toxin, for example BoNT/A, as a consequence of immunity to the clostridial toxin, for example as a result of previous exposure to the clostridial toxin, for example as a result of previous vaccination against botulism, for example vaccination using a pentavalent BoNT/A toxoid.
  • Disorders which may be appropriate to treat, particularly in the field of pediatrics, are discussed in Gordon (1999) Brain Dev 21(3), 147-51 and may include strabismus and blepherospasm, spastic cerebral palsy ,the extrapyramidal form of cerebral palsy, forms of dystonia, (generalized or focal), spasmodic torticollis and pain (for example back pain) caused by muscle spasms.
  • Inhibition of exocytosis in (ie blocking of discharges from) cholinergic sympathetic and parasympathetic terminals may be beneficial, for example in the treatment of autonomic disorders, for example focal hyperhidrosis (excessive sweating), lacrimation and salivation, particularly prominent in patients suffering from Parkinson's disease.
  • Adreno-chromaffin cells are the secretory cells of the adrenal medulla and secrete, for example, adrenaline, the effects of which closely resemble those brought about by activity of the sympathetic nervous system. Inhibition of exocytosis from adreno-chromaffin cells may be useful in the treatment of disorders or conditions in which excessive adrenaline release may be involved, for example stress. Delivery of BoNT/B or E/ into adipocytes blocks the SNARE-dependent fusion of glucose transporter 4-containing vesicles with the cell surface preventing the majority of insulin-stimulated glucose uptake (i.e. control of weight gain). Chen et al., 1997, Biochem. 36 p5719-5728). Thus, inhibition of exocytosis in these cells may be beneficial.
  • the methods or uses according to the present invention may be useful in reversing (including partially reversing, or modulating) such inhibition, for example if the inhibition of exocytosis is no longer required, or if a reduced level and or duration of inhibition is required.
  • the cell is a mammalian cell, more preferably a human or rodent cell, still more preferably a human cell.
  • SNAREs examples include SNAP-25 (synaptosomal-associated protein of 25 kDa), syndet (or SNAP-23), the VAMP (vesicle-associated membrane protein) vSNARE sub-family (which includes VAMP-1 (synaptobrevinl), VAMP-2 (synaptobrevin2) and cellubrevin) and the syntaxin tSNARE subfamily which has more than 12 isoforms with different tissue distributions as well as different cellular localizations. Syntaxin la and lb are largely neuron or neuroendocrine specific.
  • a SNARE may be capable of forming a complex with the NSF (N-ethylmaleimide-sensitive fusion protein) or a SNAP (soluble (N-ethylmaleimide-sensitive fusion protein)-attachment protein).
  • NSF N-ethylmaleimide-sensitive fusion protein
  • SNAP soluble (N-ethylmaleimide-sensitive fusion protein)-attachment protein.
  • SNAREs may contain homologous domains that form coiled-structures that may mediate interaction between SNAREs, as known to those skilled in the art.
  • SNAP-25 is present in two isoforms (a and b) in neurons (Bark (1993) JMol Biol 233, 67-76; Bark & Wilson (1994) Gene 139, 291-292).
  • the isoforms appear to arise from alternative splicing of two divergent versions of exon 5 and differ by nine amino acids and the spacing of four cysteine residues which are palmitoylated and participate in the membrane association of SNAP-25. Both forms appear to be able to support insulin secretion in HIT cells (Gonelle-Gispert et al (1999) Biochem J 339, 159-165).
  • SNAP-23 (also termed syndet) is a homologue of SNAP-25 that appears to be ubiquitously expressed and has approximately 60% amino acid identity with SNAP-25 (human SNAP-23: Ravichandran et al (1996) J Biol Chem 271, 13300-13303; mouse SNAP-23: Araki et al (1997) Biochem Biophys Res Comm 234, 257-262; Wang et al (1997) J Cell Sci 110, 505-513). SNAP-23 appears to be able to perform the function of SNAP-25 in insulin secretion when overexpressed (Sadoul et al (1997) J Cell Biol 128, 1019- 1028).
  • SNAP-25 is cleaved by BoNT/A between Glnl97 and Argl98 (numbering of full length SNAP-25). It is cleaved by BoNT/Cl between Arg 198 and Alal99 (numbering of full length SNAP-25) and by BoNT/E between Argl80 and Del 81 (numbering of full length SNAP-25). Human SNAP-23 does not appear to be cleaved by BoNT/A, BoNT/Cl or BoNT/E in vitro.
  • Rat SNAP-23 appears to be cleaved by BoNT/E and to a limited extent by BoNT/A in vitro, (see, for example, Vaidyanathan et al (1999) J Neurochem 72, 327-337 and Figure 7).
  • Syntaxin 1 is cleaved by BoNT/Cl and synaptobrevin (Sbr) by BoNT/B, ID, /F, /G and TeTx [reviewed by Pellizzari, R., Rossetto, O., Schiavo, G., and Montecucco, C. (1999) Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses. Philos. Trails. R. Soc. Lond. B. Biol Sci. 354, 259-68].
  • Human VAMP-2 (synaptobrevin2) is cleaved between Gln76 and Phe77 by TeTx (see, for example Shiavo et al (1992) EMBO J 11, 3577). Cellubrevin is also cleaved by TeTx.
  • the said inhibitory SNARE may be a fragment of SNAP-25, synaptobrevin or syntaxinl, which terms are defined above. It is preferred that it is a fragment of SNAP-25, still more preferably a fragment of SNAP-25 derivable by cleavage of SNAP-25 by BoNT/A or BoNT/Cl, for example derivable by cleavage of SNAP-25 or a variant thereof by BoNT/A between residues 197 and 198 of full length SNAP-25.
  • the fragment may be synthesised, for example using techniques of molecular biology or synthetic peptide synthesis, without need for cleavage by a clostridial toxin, for example BoNT/A.
  • the said inhibitory SNARE is a fragment of SNAP-25 (or a variant thereof) wherein residues corresponding to residues 198 (or less preferably 199) to 206 of full length mouse SNAP-25 are not present.
  • the fragment may consist of residues identical to residues 1 or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 140, 160, 170 or 180, preferably between 1 and about 140, to 197 of full length SNAP-25 or a variant thereof.
  • the fragment may consist of residues identical to residues 1 or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 140, 160, 170 or 180, preferably between 1 and about 140, to 198, 199, 200 or 201 of full length SNAP-25 or a variant thereof.
  • Such fragments of SNAP-25 may be capable of inhibiting SNARE-dependent exocytosis, as described in WO01/81038.
  • the fragment is not one that consists of residues equivalent to or identical to residues 1 or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 140, to 180 of full length SNAP-25 (SNAP- 25(1-180)); such a fragment may be derivable by cleavage of SNAP-25 by BoNT/E.
  • Such a fragment may be capable of inhibiting SNARE-dependent exocytosis (see Figure 8 and as described in WOO 1/18038), but is not considered to be as persistent an inhibitor of exocytosis as SNAP-25(1-197); thus, inhibition arising from a fragment derivable by cleavage of SNAP-25 by BoNT/A may be reversed/reduced by cleavage of the fragment by BoNT/E, which gives a transient (full but rapidly reversed) block.
  • the said inhibitory SNARE may be capable of inhibiting exocytosis in a cell capable of performing .SNARE-dependent exocytosis, (in vitro or in vivo), preferably a cell of the same or similar type to the said cell in a patient, by at least (in order of preference) 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 or 90% compared to a control cell to which the said inhibitory SNARE is not supplied. Cell lines or cells which may.
  • an inhibitory SNARE may include adreno- chromaffin cells (see for example WOOl/18038 and O'SuUivan et al (1999)), RIN (rat) and HIT (hamster) insulin-secreting cells (discussed in Gonelle-Gispert et ⁇ (1999)).
  • Example 1 describe an in vivo system that may be used for assessing the effect of the inhibitory treatment described above and methods of reversal of inhibition. The method involves repeated in vivo imaging of nerve terminals and measurements of depolarisation-evoked endo- and exo-cytosis.
  • the inhibitory SNARE is preferably a fragment derivable by cleavage of synaptosomal-associated polypeptide of 25 kDa (SNAP-25) or a variant thereof by BoNT/A.
  • the inhibitory SNARE consists of residues identical to residues 1 to 197 of full length SNAP-25 or a variant thereof (SNAP-25 A).
  • BoNT/E-truncated SNAP-25 does not support exocytosis and significantly inhibits exocytosis (see Figure 8), but for a much shorter period than BoNT/A- or BoNT/Cl -truncated SNAP-25. Its presence may promote intracellular movement and/or degradation of SNAP-25 / , as discussed in Example 1.
  • Cleavage of a proportion of SNAP-25 or SNAP-25 A molecules by BoNT/E therefore appears to be sufficient for the reversal of inhibition caused by the presence of SNAP-25 ⁇ - Supplying SNAP-25E molecules to a cell (for example by expression of the SNAP-25j ⁇ in the cell) may also promote intracellular movement and/or degradation of SNAP-25 ⁇ . and may therefore also be useful in the reversal of inhibition caused by the presence of SNAP-25A-
  • Example 1 Suitable methods for detecting and identifying SNAP-25 and fragments derivable from SNAP-25 in cells are described in Example 1.
  • the agent is capable of causing cleavage of the inhibitory SNARE.
  • the product(s) of the cleavage preferably cause significantly less persistent inhibition than the uncleaved inhibitory SNARE.
  • the agent may comprise a clostridial toxin, for example a botulinum toxin, by which the inhibitory SNARE is capable of being cleaved.
  • the inhibitory SNARE is capable of being cleaved by BoNT/E and the agent comprises BoNT/E (or at least the catalytic portion ie light chain of BoNT/E).
  • the inhibitory SNARE is not resistant to cleavage by BoNT/E ie is not a BoNT/E-resistant inhibitory SNARE.
  • variants of SNAP-25 that are resistant to BoNT/E include variants in which the residue equivalent to residue 180 and/or the residue equivalent to residue 181 of full length SNAP-25 (for example full length mouse SNAP-25) are replaced by a residue other than Arg or a residue other than He, respectively.
  • Ilel ⁇ l may be replaced by Phe, Gly, Ser or Asn. Replacement by Val may also result in a small increase in resistance to BoNT/E cleavage (Vaidyanathan et al (1999) JNeurochem 72, 327-337).
  • Argl76, As ⁇ l79 and/or Metl82 may further or alternatively be mutated,. for example to Pro 176, Lysl79 and/or Thrl82 (see Gonelle-Gispert et al (1999).
  • variants are not preferred.
  • BoNT/E is included any variant, fragment, derivative or fusion of naturally occurring BoNT/E that retains the catalytic activity of BoNT/E, particularly the ability to cleave SNAP-25 in the same place as naturally occurring BoNT/E. It is preferred that the BoNT/E retains the cell-binding specificity of naturally occurring BoNT/E (ie may retain the heavy chain of naturally occuring BoNT/E), as well known to those skilled in the art, for example when treating a patient with BoNT/A or BoNT/C poisoning.
  • BoNT/E When treating a patient in which exocytosis has been inhibited in a cell type to which botulinum toxins do not bind, for example by supply (for example expression) of an inhibitory SNARE to the cell, it may be desirable for the BoNT/E to be targeted to (and taken up by, or expressed inside) that cell type, using methods known to those skilled in the art: in this case, it is not necessary for the BoNT/E to retain the cell binding specificity of naturally occuring BoNT/E. Thus, the light chain of BoNT/E may be retained but not the heavy chain. It will be appreciated that BoNT/E may be expressed in cells as an alternative to delivering the actual polypeptide.
  • the agent may comprise a polynucleotide encoding and capable of expressing BoNT/E, as defined above, for example encoding at least the catalytic portion of BoNT/E.
  • Suitable delivery vehicles are described in WOOl/18038, for example adenoviral vectors with cholinergic-specific promoters may be used.
  • a toxin-resistant, for example BoNT/E-resistant, SNARE may typically be a non-naturally occurring SNARE, ie a synthetic, protease-resistant variant of a naturally occurring SNARE that is capable of being cleaved by the said clostridial toxin.
  • a toxin-resistant SNARE may be a naturally occurring toxin-resistant SNARE.
  • SNAP-23 for example is a naturally occurring toxin-resistant SNARE (at least in certain species); an inhibitory SNARE derivable therefrom is therefore not preferred when the active ingredient of the agent is BoNT/E.
  • a toxin-resistant SNARE is included the meaning that the toxin-resistant SNARE is cleaved by the relevant clostridial toxin to a lesser extent than a SNARE that is cleaved by the said clostridial toxin (for example SNAP-25 for BoNT/A, BoNT/C or BoNT/E; synaptobrevin for BoNT/B, D, F or G and TeTx; syntaxin for BoNT/C).
  • cleaved to a lesser extent is included the meaning that at least about 1.2, 1.5, 2, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000, 30000 or 40000 more of the said clostridial toxin is required to cleave 50% of the said toxin resistant SNARE than is required to cleave 50% of full length human SNAP-25 (for BoNT/A, BoNT/C or BoNT/E) or full length human synaptobrevin (for BoNT/B, D, F or G and TeTx) or full length human syntaxin 1 (for BoNT/C) under the same conditions, for example the conditions employed in the experiments summarised in Table 1 and described in Example 1 of WOOl/18038.
  • the agent which is capable of reducing the amount of the inhibitory SNARE in the cell and/or altering the location of the inhibitory SNARE in the cell may be SNAP-25j7 or a polynucleotide encoding and capable of expressing SNAP-25J7.
  • the presence of SNAP-25j? in the cell may promote removal and/or relocation of the inhibitory SNARE (for example SNAP-25 ⁇ or SNAP-25ci) and may therefore be useful in reducing persistent inhibition of exocytosis in the cell.
  • SNAP-25J? or a polynucleotide encoding SNAP-25J? may provide advantages over using BoNT/E, because the former agents may be usable in a wider range of cell types than BoNT/E. In addition, the former agents may be easier to control in use and more reliable.
  • SNARE that is capable of functioning in SNARE-dependent exocytosis (functional SNARE) to the patient, particularly to an affected cell of the patient, as described in WOOl/18038. This may speed recovery of exocytosis in the cell.
  • an inhibitory SNAP-25 molecule for example SNAP-25 ⁇
  • the supply of the, for example, full-length SNAP-25 may be by administering the full-length SNAP-25 to the cell or by expressing the full-length SNAP-25 (SNAP-25wt) in the cell, for example from a recombinant polynucleotide.
  • the inhibitory SNARE When the inhibitory SNARE was formed in the cell as a result of exposure of the cell to a clostridial toxin, for example BoNT/A or BoNT/Cl, it may be desirable for the SNARE that is capable of functioning in SNARE- dependent exocytosis that is supplied to the cell to be resistant to cleavage by the said clostridial toxin, for example BoNT/A or BoNT/Cl.
  • the said functional SNARE is also resistant to cleavage by the agent.
  • a patient or cell with BoNT/A poisoning and/or in which SNAP-25A is present may be treated using BoNT/E as the agent (to cleave the to the non-inhibitory SNAP-25 ⁇ ), and by administering or expressing SNAP-25 that is resistant to both BoNT/A and BoNT/E.
  • a SNARE which is capable of inhibiting the clostridial toxin (toxin- inhibitory SNARE), as discussed in WOOl/18038 may also usefully be supplied to the cell as indicated above.
  • the toxin-resistant SNARE or toxin-inhibitory SNARE may be a variant, fragment, derivative or fusion of a naturally occurring SNARE with the required or preferred properties (for example in relation to their ability to support SNARE-dependent exocytosis) as discussed in WOOl/18038.
  • variants of a polypeptide for example of SNAP-25, syntaxin 1 or synaptobrevin
  • insertions, deletions and substitutions either conservative or non-conservative.
  • variants of the polypeptide where such changes do not substantially alter the activity of the said polypeptide, for example the ability of the SNAP-25, syntaxin 1 or synaptobrevin to participate in a ternary complex comprising SNAP-25, syntaxin 1 or synaptobrevin (or homologues thereof) which is capable of supporting exocytosis, for example as described in WOOl/18038.
  • substitutions is intended combinations , such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • residue equivalent to a particular residue, for example the residue Argl98 of full-length SNAP-25, for example mouse or human SNAP-25, is included the meaning that the amino acid residue occupies a position in the secondary or three dimensional structure of a native polypeptide, for example a SNAP-25 homologue or variant, corresponding to the position occupied by the said particular residue, for example Argl98, in the native secondary or three dimensional structure of full-length SNAP-25. It will be appreciated that Argl98 of full-length SNAP-25 is located towards the C- terminus of the polypeptide.
  • the residue equivalent to a particular residue may be identified by alignment of the sequence of the polypeptide with that of full-length SNAP-25 in such a way as to maximise the match between the sequences.
  • the alignment may be carried out by visual inspection and/or by the use of suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group, which will also allow the percent identity of the polypeptides to be calculated.
  • the Align program (Pearson (1994) in: Methods in Molecular Biology, Computer Analysis of Sequence Data, Part II (Griffin, AM and Griffin, HG eds) pp 365-389, Humana Press, Clifton).
  • residues identified in this manner are also "equivalent residues”.
  • the three-letter and one-letter amino acid code of the IUPAC-IUB Biochemical Nomenclature Commission is used herein.
  • the sequence of polypeptides are given N-terminal to C-terminal as is conventional.
  • Xaa represents any amino acid.
  • the amino acids are L- amino acids; in particular it is preferred that the amino acid residues immediately flanking (such as those within 10 to 20 residues) of the clostridial toxin cleavage site consists of L-amino acid residues but they may be D-amino acid residues.
  • the patient may have botulism, in particular botulism caused by, or by a strain producing BoNT/A or BoNT/Cl (or less preferably BoNT/E, BoNT/D, BoNT/F or BoNT/G), preferably BoNT/A.
  • BoNT/A or BoNT/Cl or less preferably BoNT/E, BoNT/D, BoNT/F or BoNT/G
  • BoNT/A BoNT/A
  • Types of botulism and methods of diagnosing botulism are known to those skilled in the art and are summarised above.
  • the patient may be an infant, for example an infant with the symptoms of a "floppy baby", that has been diagnosed as having botulism, as described above and, for example, in Greve et al (1993) Monatsschr Kinderheilkd 141(1), 33-35; Mid ⁇ ra (1979) Rev Infect Dis 1(4), 652-655; Puig de Centorbi (1998) Monasham, 652-655; Pickett (1982) Muscle Nerve 5(9S), S26-27.
  • the method of diagnosing botulism preferably allows the type of botulinum toxin that is responsible for the poisoning to be determined.
  • the method of treatment of the invention described above may further comprise the steps of determining the type of the said clostridial, for example botulinum, toxin from which the patient is suffering and of selecting an appropriate agent (in the appropriate aspect of the invention) for use in the treatment.
  • the type of the clostridial toxin from which the patient is suffering from poisoned by may be determined.
  • the patient is suffering from poisoned by BoNT/A or BoNT/C 1 , most preferably BoNT/A.
  • Toxin may be identified, for example, from stool specimens, as reviewed, for example, in Pickett (1982) Muscle Nerve 5(9S), S26-27 and Cherington (1998) Muscle Nerve 21(6), 701-710.
  • BoNT/A is the toxin type generally utilised in treating neuromuscular conditions and is available commercially from several sources; for example from Porton Products Ltd, UK under the trade name "Dysport”TM, and from Allergan, Inc., Irvine, California under the trade name "BOTOX”TM.
  • a further aspect of the invention provides a kit of parts comprising (1) means for determining the type of clostridial, for example botulinum, toxin from which a patient is suffering or means for determining that a patient is suffering from a particular type of clostridal, for example botulinum, toxin (preferably BoNT/A or BoNT/Cl) and (2) an agent as defined in relation to previous aspects of the invention, for example comprising BoNT/E, or comprising SNAP-25E or a polynucleotide encoding SNAP-25j7.
  • a further aspect of the invention provides a kit of parts comprising (1) an agent as defined in relation to previous aspects of the invention, for example comprising BoNT/E, and (2) an inhibitor of the (or a) clostridial, for example botulinum, toxin from which the patient is suffering or the cell has been exposed to.
  • the inhibitor may preferably inhibit BoNT/A or BoNT/Cl, for example when the agent comprises BoNT/E.
  • the inhibitor may be a toxin-inhibitory SNARE or recombinant polynucleotide capable of expressing said toxin-inhibitory SNARE, as described in WOOl/18038.
  • the kit may further comprise means for determining the type of clostridial, for example botulinum, toxin from which a patient is suffering or means for determining that a patient is suffering from a particular type of clostridal, for example botulinum, toxin, as described above.
  • a further aspect of the invention provides a kit of parts comprising an inhibitory SNARE or polynucleotide encoding an inhibitory SNARE, and an agent capable of cleaving the inhibitory SNARE, as defined in relation to the fifth and sixth aspects of the invention.
  • the kit thus provides means for inhibiting exocytosis, and means for reversing such inhibition.
  • the patient is a human.
  • the patient may be a non-human mammal, for example a domesticated animal, for example a rodent (for example a mouse or a rat) or domesticated mammal, for example a horse or dog. It will be appreciated that many types of live stock or domesticated animals are susceptible to botulism.
  • the high level of sequence identity between equivalent SNAREs, for example SNAP-25s, from different animals, for example mammals may mean that an inhibitory SNARE that is capable of inhibiting exocytosis in cells of one type of animal, for example mammal, may also be capable of inhibiting exocytosis in cells of a different type of animal and the methods/uses of the invention may therefore be useful in relation to treating both types of animal which have been treated using that inhibitory SNARE.
  • the patient to be treated is administered an effective amount of the said agent.
  • effective amount we include an amount sufficient to produce a clinically useful or significant reduction in any symptoms arising from the inhibition of exocytosis, for example symptoms of poisoning by a clostridial toxin, for example BoNT/ A, in the said patient.
  • the effective amount may produce an increase in exocytosis in the said cell.
  • the agent comprises BoNT/E
  • the time elapsed before clinically useful or significant reduction in any symptoms are apparent may depend on the dose of BoNT/E used, but may be within about 5 to 10 days. Generally, if more BoNT/E is used, the shorter the recovery time may be. A shorter recovery time may be achieved if the agent comprises SNAP-25j? or a polynucleotide encoding SNAP-25E rather than BoNT/E.
  • the methods or constructs of the invention may be evaluated in, for example, dissociated primary neuronal cell cultures, motor neurons, chromaffin cells and/or nerve-muscle co-cultures, as known to those skilled in the art, before evaluation in whole animals.
  • the methods described in de Pavia et al (1999) Proc Natl Acad Sci USA 96, 3200-3205 may also be used in the evaluation of the methods or constructs of the invention.
  • the BoNT/E (or other agent, as appropriate) may be injected at the site of the affected cells.
  • the BoNT/E (or other agent) may be injected at the same site as the moiety that was used to produce the deliberate inhibition, or may otherwise be administered in the same manner.
  • the methods may be used in conjunction with other methods, for example administration of neutralising antibodies and/or BoNT inhibitors.
  • the patient may be assessed in order to determine the stage of poisoning in order to decide on the most appropriate combination and/or order of treatment.
  • the order may be administration of neutralising antibodies; administration of BoNT inhibitors; followed by rescue of regulated exocytosis by replacement with full length protein (as described in WOOl/18038) and or methods as described herein, for example supply of BoNT/E or SNAP-25 E .
  • Suitable vectors and delivery systems including systems in which expression of the encoded polypeptide is under the control of an inducible promoter, are described in, for example WOOl/18038.
  • a further aspect of the invention provides the use of BoNT/E or a polynucleotide encoding and capable of expressing BoNT/E, or SNAP-25p? or a polynucleotide encoding and capable of expressing SNAP-25TH, in the manufacture of a medicament for the treatment of a patient in need of short duration inhibition of exocytosis in a cell of the patient, wherein the medicament does not comprise BoNT/A, B, C, F or G.
  • a further aspect of the invention provides a method for treating a patient in need of short duration inhibition of exocytosis in a cell of the patient, wherein the patient is administered BoNT/E and is not administered BoNT/A, B, C, F or G.
  • a further aspect of the invention provides a method for treating a patient in need of short duration inhibition of exocytosis in a cell of the patient, wherein the patient is administered a recombinant polynucleotide encoding and capable of expressing BoNT/E; or SNAP-25TH; or a recombinant polynucleotide encoding and capable of expressing SNAP-25JH . It is preferred that the patient is not administered BoNT/A, B, C, F or G.
  • BoNT/E or SNAP-25j ⁇ it is desirable for expression of BoNT/E or SNAP-25j ⁇ to be transient or to be capable of being controlled temporally, for example to be under the control of an inducible promoter, as well known to those skilled in the art and as discussed in WOOl/18038.
  • the inducer molecule may be suitable for oral administration, and is preferably administered in this way.
  • a further aspect of the invention provides a recombinant polynucleotide encoding and capable of expressing BoNT/E; SNAP-25E; or a recombinant polynucleotide encoding and capable of expressing SNAP-25 ⁇ , for use in medicine.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant polynucleotide encoding and capable of expressing BoNT/E; SNAP-25]?; or a recombinant polynucleotide encoding and capable of expressing SNAP-25 ⁇ and a pharmaceutically acceptable excipient.
  • Suitable excipients and formulations will be well know to those skilled in the art and are described in, for example, WOOl/18038, and may include sterile saline solution or distilled water which is pyrogen free.
  • a further aspect of the invention provides a gene therapy construct comprising a recombinant polynucleotide encoding and capable of expressing BoNT/E or a recombinant polynucleotide encoding and capable of expressing SNAP-25 ⁇ .
  • suitable vectors, gene therapy constructs and delivery systems which may be adapted in relation to the present invention, including systems in which expression of the encoded polypeptide is under the control of an inducible promoter, are described in, for example WOO 1/18038.
  • BoNT/E differs from other botulinum toxins, for example BoNT/F, in that it does not elicit nerve sprouts and therefore is less likely than other botulinum toxins to cause permanent damage to the treated cells, as shown in Example 1.
  • Use of BoNT/E (or the rapidly degraded SNAP-25]g) is therefore highly preferred in situations where short-term muscle weakness or immobilisation, followed by full recovery of muscle strength, is required.
  • the patient may be in need of inhibition of exocytosis of less than 14 days' duration, still more preferably in need of inhibition of exocytosis of less than 7, 6 or 5 days' duration.
  • the patient may be in need of inhibition of muscle contraction.
  • the patient may be in need of temporary immobilisation of a joint or prevention of muscle contractions prior to, during or after surgery, treatment of joint dislocation, alleviation of muscle spasm, treatment of tendons or ligaments, treatment of scoliosis or spasm of sphincter muscles.
  • the patient may be in need of relief of pain arising from muscle contractions.
  • the invention provides a method for inhibiting muscle contraction, relieving pain, temporarily immobilising a joint or preventing muscle contractions prior to, during or after surgery, treating joint dislocation, alleviating muscle spasm, treating tendons or ligaments, treating scoliosis or spasm of sphincter muscles, wherein the patient is administered BoNT/E or a polynucleotide encoding and capable of expressing BoNT/E, or SNAP- 25JH or a polynucleotide encoding and capable of expressing SNAP-25TH, and wherein the patient is in need of short duration of the effects of treatment and is not administered BoNT/A, B, C, F or G.
  • the invention further provides the use of BoNT/E or a polynucleotide encoding and capable of expressing BoNT/E, or SNAP-25JH or a polynucleotide encoding and capable of expressing SNAP-25j7 in the manufacture of a medicament for inhibiting muscle contraction, relieving pain, temporarily immobilising a joint or preventing muscle contractions prior to, during or after surgery, treating joint dislocation, alleviating muscle spasm, treating tendons or ligaments, treating scoliosis or spasm of sphincter muscles, wherein the patient in need of short duration of the effects of treatment and is not administered BoNT/A, B, C, F or G.
  • BoNT/E or SNAP ⁇ 25j? it is desirable for expression of BoNT/E or SNAP ⁇ 25j? to be transient or to be capable of being controlled temporally, for example to be under the control of an inducible promoter, as well known to those skilled in the art and discussed in WOOl/18038. It is preferred that the inducible promoter is controlled by an inducer molecule which is suitable for oral administration.
  • the patient may be suffering from a sports injury or muscle cramps.
  • the patient may be suffering from a tension headache.
  • Smooth muscle disorders that may be treated include spasms of the sphincter of the cardiovascular arteriole, gastrointestinal system, urinary or gall bladder or rectum.
  • the patient may be undergoing or about to undergo total joint replacement, treatment of compound fractures, treatment of joint infections or dislocations.
  • BoNT/E BoNT/E
  • BoNT/A BoNT/A in the same muscle group
  • the patient may alternatively be in need of short duration treatment of conditions such as cholinergic controlled secretions including excessive sweating, lacrimation, salivation and mucus secretions. This may be useful for a performer such as an actor, musician or public speaker during a performance or presentation.
  • Botulinum toxin E may exist in a dichain form or a single chain (un-nicked) form.
  • the single chain form is less active than the dichain form but may be converted to the corresponding dichain form by nicking with a protease, for example trypsin. Both the single and the dichain form may be useful in relation to the present invention.
  • An appropriate activatable recombinant neurotoxin as described in WOOl/14570 may be used (ie one which has BoNT/E catalytic activity).
  • BoNT/E is included any variant, fragment, derivative or fusion of naturally occurring BoNT/E that retains the catalytic activity of BoNT/E, particularly the ability to cleave SNAP-25 in the same place as naturally occurring BoNT/E. It is preferred that the BoNT/E retains the cell-binding specificity of naturally occuring BoNT/E, as well known to those skilled in the art. When the BoNT/E is expressed in the desired cell, it is not necessary for the cell-binding portion of the wt BoNT/E to be expressed.
  • Botulinum toxins may be obtained commercially or by establishing and growing cultures of appropriate C botulinum strains in a fermenter and then harvesting and purifying the fermented mixture in accordance with known techniques.
  • Commercial sources of BoNT/E are mentioned in Example 1.
  • the toxin (or in relation to the fifth and sixth aspects of the invention, other agent) is administered by means of intramuscular injection (when appropriate for the condition to be treated) directly into a local area such as a spastic muscle, preferably in the region of the neuromuscular junction, although alternative types of administration (for example subcutaneous injection), which can deliver the toxin directly to the affected region, may be employed where appropriate.
  • the toxin may be presented as a sterile pyrogen-free aqueous solution or dispersion and as a sterile powder for reconstitution into a sterile solution or dispersion, as known to those skilled in the art.
  • Tonicity adjusting agents such as sodium chloride, glycerol and various sugars may be added, as known to those skilled in the art.
  • Formulations suitable for use with other botulinum toxins for example BoNT/A, as known to those skilled in the art, may be suitable for use with BoNT/E.
  • Suitable formulations may be described in, for example WO95/17904 and W094/28923.
  • Preferred unit dosage formulations are those containing a daily dose, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
  • the dose of BoNT/E, SNAP-25E or polynucleotide (or where appropriate, other agent, as defined above) administered to the patient may depend upon the severity and extent of the botulinum toxin poisoning or inhibition of exocytosis to be treated. For example, it may depend on the number of muscle groups (or other cell types) requiring treatment, the age and size of the patient, and the type of toxin or inhibitory SNARE causing the poisoning/inhibition. Examples of useful doses and of methods of assessing useful doses are described in Example 1.
  • the potency of the toxin may be expressed as described in Example 1 (ie equivalent dose (ED) in relation to generation of maximal loss of toe spread reflex in mice without other obvious symptoms of botulism). Less preferably, the potency of the toxin may be expressed as a multiple of the LD50 value for the mouse, one unit (U) of toxin being defined as being the equivalent amoung of toxin that kills 50% of a group of 18 to 20 female Swiss-Webster mice, weighing about 20 grams each, within 4 days.
  • ED equivalent dose
  • U the equivalent amoung of toxin that kills 50% of a group of 18 to 20 female Swiss-Webster mice, weighing about 20 grams each, within 4 days.
  • a dose of between about 2 to 0.1, preferably 1 to 0.2, still more preferably between about 0.7 and 0.3 ED of BoNT/E may be useful in reversing paralysis of mouse muscle caused by about 0.5 ED of BoNT/A, measured as described in Example 1. Larger doses may be required in larger animals or target tissues.
  • the BoNT/E (or other agent) may be administered in a single or multiple doses. The quantity administered and the frequency of administration will be at the discretion of the responsible physician and will depend on the response of the patient to the treatment.
  • the anatomy of the muscle group is considered carefully, the aim being to inject the area with the highest concentration of neuromuscular junctions, if known, or the area previously injected, for example with BoNT/A).
  • the position of the needle in the muscle may be confirmed by putting the muscle through its range of motion and observing the resultant motion of the needle end.
  • General anaesthesia, local anaesthesia and sedation are used according to the age of the patient, the number of sites to be injected, and the particular needs of the patient. More than one injection and/or sites of injection may be necessary in order to achieve the desired result. It may be necessary (depending on the required site of injection) to use a fine, hollow, TeflonTM-coated needle, guided by electromyography. Suitable administration techniques are described in, for example W095/17904 and W094/28923.
  • the improvement in the patient's condition may be assessed subjectively and/or objectively.
  • Fig. 1 Time courses for recovery from neuromuscular paralysis induced by BoNT E or F are faster than that seen following type A injection into mouse leg muscles: BoNT E shortens the duration of the action of type A but not F toxin.
  • BoNT E After intramuscular injection of one ED of BoNT/A (•), BoNT/E (O) or BoNT/F (T) into the right hind-leg of mice, loss of neuromuscular transmission was assessed by determining the TSR score (5 is maximum paralysis).
  • Fig. 2 The extensive remodeling and switching in synaptic activity between the original nerve endings and their sprouts following paralysis with BoNT/A was less pronounced with BoNT/F poisoning and not detectable after type E.
  • injection of 0.05 ED of BoNT/A into mouse sternomastoid muscle was shown to result in a loss of the ability of the original endplates (stained with 4-di-2-ASP, green in A, filled bars in D) to exo-endocytose FM1-43 (red) upon stimulation with 60 mM K+ (A and D) and an outgrowth of sprouts (arrows) capable of stimulated uptake of this dye (A and empty bars in D).
  • Fig. 3 Persistence of SNAP-25 A in BoNT/A-treated murine motor nerve terminals.
  • Control and BoNT/A-treated endplates were dual-labeled with rhodamine-conjugated ⁇ -bungarotoxin and anti-SNAP-25A antibody, followed by FITC-conjugated secondary IgGs; fluorescent images were recorded by confocal microscopy, as detailed in Materials and Methods.
  • Fig. 4 Distribution of total SNAP-25 at the NMJ during BoNT/A- induced paralysis: disappearance of the sprouts following subsequent injection of BoNT/E.
  • Control and BoNT/A-treated endplates were dual- labeled with rhodamine-conjugated ⁇ -bungarotoxin and anti-SNAP-25pL followed by FITC-conjugated secondary IgGs; the images were recorded by confocal microscopy.
  • SNAP-25pL was detected in nerve terminals where it co-localized with areas occupied by the nAChR.
  • BoNT/A-treated preparations some immunostaining was detected beyond the boundaries of nAChR, in sprouts (d6, d20; see arrows).
  • Fig. 6 Fate of SNAP-25 A and SNAP25 E at the NMJ upon sequential injection of BoNT/E 3 days after type A.
  • Control and BoNT/A-treated mouse sternomastoid followed (after 3 days) in the latter case by injection of BoNT/E were dual-labeled with rhodamine-conjugated ⁇ -bungarotoxin and either anti-SNAP-25A or ⁇ SNAP25 ⁇ followed by FITC-conjugated secondary IgGs.
  • Confocal microscopy revealed that SNAP25A was detectable up to 11 days after BoNT/E injection in a few branches of the motor nerve terminals and pre-terminal axons; this staining was no longer seen 4 days later (dl5).
  • Fig. 7 Distribution and quantitation of SNAP-25 FL and SNAP-25 A immunostaining during BoNT/A treatment alone or with a subsequent injection of BoNT/E 7 days later.
  • Nerve terminals in mouse sternomastoid were treated with BoNT/A alone (A, B) and additionally with BoNT/E 7 days later (C), as in Fig. 6, and then stained with IgGs specific for SNAP-25pL ( A ) or SNAP-25A (B, C); all the samples were labeled with rhodamine-conjugated ⁇ -bungarotoxin, followed by FITC-conjugated secondary antibodies.
  • Figure 8 BoNT/A or E truncated SNAP-25, shown to be expressed in CHO cells, inhibited evoked secretion in intact chromaffin cells.
  • A CHO cells, that lack SNAP-25, were transfected with the pcDNAl.l/Amp vector incorporating the specified SNAP-25-R198T gene using SuperfectTM reagent, as described in O'SuUivan et al 1999.
  • SNAP-25(1-197) is SNAP-25A; SNAP-25(1-180) is SNAP-25 ⁇ .
  • Example 1 Recovery of synaptic activity to mouse endplates paralysed by botulinum toxin type A is hastened by the short-acting type E toxin due to the removal of truncated SNAP-25
  • Quantal neurotransmitter release is inhibited selectively by seven serotypes (A-G) of botulinum neurotoxin (BoNT) whose Zn2+-dependent protease cleaves SNARE proteins that are essential for this fundamental process of Ca2+-regulated exocytosis.
  • BoNT/A and /E proteolyse SNAP-25 at neighboring bonds their blockade of acetylcholine release from mouse motor nerves following local injection caused flaccid muscle paralysis for very different durations (30 and 5 days, respectively).
  • BoNT/E injection into mouse muscle was shown to inhibit depolarisation- dependent uptake of the dye, FM1-43, but the vesicle recycling resumed after 5 days and there was an absence of detectable nerve sprouting.
  • neuroparalysis resulting from BoNT/A or /F induced the appearance of nerve sprouts, that exhibited FM1-43 uptake, and these were eliminated when the parental terminals recovered functionality.
  • the extent and life-time of the sprouts are related reciprocally to the duration of neuromuscular paralysis by the various toxins.
  • This defective protein was found to be translocated from the presynaptic membrane and removed from the terminal following co-injection of the other SNAP-25-targeting toxin, BoNT/E; such dis-inhibition of the trafficking of the SNAP-25A would allow replenishment of the intact active protein and could overcome the proteolytic action of any BoNT/A activity remaining.
  • botulinum neurotoxin/type A, E, BoNT/A, E effective dose, ED; nicotinic acetylcholine receptors, nAChRs; cleaved products of BoNT/A and /E, SNAP-25A and SNAP-25E; PBS, phosphate-buffered saline; TSR, toe spread reflex; 4-di-2-ASP, 4-(4-diethyl aminostyryl)-N- methylpyridinium iodide; FM1-43, N-(3-triethyl ammonium propyl)-4-(4- (dibutylamino)styryl) pyridinium dibromide.
  • BoNT/A, E and F are also available from other sources, for example Sigma-Aldrich Company Ltd, Fancy Road, Poole, Dorset, BH12 4QH, UK (catalogue numbers B8776, B6528 and B9152 respectively). It should be noted that the ED determined for each toxin gave maximal paralysis of the extensor digitorus longus muscle in the absence of any other symptoms of botulism in the mice. The mice were allowed to recover and the loss of toe spread reflex (TSR) scored from 0 to 5 (where 5 is a complete absence of TSR) following their examination twice daily (Pockett and Gavin, 1985).
  • TSR loss of toe spread reflex
  • Nerve endings were stained for 5 min with either 5 ⁇ M 4-(4-diethyl aminostyryl)-N-methylpyridinium iodide (4-di-2- ASP; Molecular Probes) alone in aerated Krebs-Ringer medium ([mM] NaCl, 118; KC1, 4.69; MgS04, 1.18; KH P0 , 1.18; glucose, 11.7; NaHC03, 23.8; CaCl2, 2.52, pH 7.4; (de Paiva et al, 1999), or in Krebs-Ringer with elevated K + concentration (60 mM KC1 and 58 mM NaCl) containing both 5 ⁇ M 4-di-2- ASP and 4 ⁇ M N-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43; Molecular Probes).
  • each mouse was positioned under a Zeiss Axioskop fixed-stage microscope equipped with epifluorescence. Staining with 4-di-2-ASP was visualized with a FITC-type narrow band-pass filter block (450-490 nm excitation ⁇ , 515-565 nm emission ⁇ ) with detection of labeling with 4-di-2-ASP and FM1-43 being achieved using the above filter and a long-pass rhodarnine-type block (524-556 nm excitation ⁇ , > 590 nm emission ⁇ ), respectively.
  • a FITC-type narrow band-pass filter block 450-490 nm excitation ⁇ , 515-565 nm emission ⁇
  • detection of labeling with 4-di-2-ASP and FM1-43 being achieved using the above filter and a long-pass rhodarnine-type block (524-556 nm excitation ⁇ , > 590 nm emission ⁇ ), respectively.
  • nAChRs postsynaptic nicotinic acetylcholine receptors
  • Samples were imaged with a laser scanning microscope (Zeiss 510) mounted on an upright microscope (Axioplan-2 Zeiss) and operated with the manufacturer's software (LSM 510 version 1.49.44) running on Windows NT 4.0 operating system (Microsoft, U.S.A.).
  • LSM 510 version 1.49.44 the manufacturer's software
  • Windows NT 4.0 operating system Microsoft, U.S.A.
  • BoNT/A poisoning are the original endplates unable to undergo neurotransmitter release for such a prolonged period whereas the other SNAP-25-targetted toxin, serotype E, causes a contrasting short-lived paralysis?
  • the action of BoNT/E was studied in conjunction with BoNT/A. If the durations of the paralysis were solely dependent on the lifetime of the neurotoxins within the nerve terminals, then co- administering both serotypes should give a recovery profile dependent on the longer-lasting BoNT/A.
  • BoNT/E (0.5 of the ED) was injected 3 days after an initial administration of BoNT/A (0.5 of its ED); this should allow adequate time for BoNT/A to establish its paralysis pattern.
  • a full recovery from this procedure was observed at ⁇ day 13 after the second injection (Fig. 1C), corresponding to the time course of recovery following the co- injection (Fig. IB). It is, therefore, unlikely that the recovery from the sequential or co-injection of BoNT/E with BoNT/A is due to an impaired uptake of the latter. On the contrary, it indicates BoNT/E speeds up the molecular events underlying the recovery of nerve-induced muscle twitch (Eleopra et al, 1998).
  • the time point for sprout elimination can be manipulated by over-riding the prolonged paralysis existence of SNAP-25A in BoNT/A- paralysed preparations with a delayed injection of another SNAP-25-targetting serotype, BoNT/E, thereby, resulting in a shortening of the recovery process and, consequently, an earlier induction of sprout elimination (see Fig. 4 and 5).
  • BoNT/A long-lasting blockade of release by BoNT/A could only be affected by: (i) an extended life-time of BoNT/A protease activity within the motor neurons (Keller et al, 1999) and neuro-endocrine cells (O'SuUivan et al, 1999) and/or (ii) impairment of SNAP-25pL incorporation at the release sites due to persistence of SNAP-25A (one possibility mentioned by Eleopra et al, 1998).
  • BoNT/A and /E Co-injection of BoNT/A and /E was shown to shorten the paralysis time expected for type-A intoxication, which seems to preclude persistence of adequate activity of the BoNT/A toxin within the original endplate (Eleopra et al, 1998). Indeed, if BoNT/A-proteolytic activity was chronically persistent when compared to BoNT/E, a much longer paralysis should have been reported. In sharp contrast, the lifetime of BoNT/A was found to exceed by far that of BoNT/E in a spinal cord neuronal culture treated sequentially with 0.4 pM BoNT/A followed 3 days later with 250 pM (Keller et al, 1999).
  • SNAP-25A seems to be a major hindrance to recovery of neurotransmitter at the original endplates and this, probably, results from a competition between SNAP-25A and SNAP-25pL f° r SNARE binding partners at release sites.
  • BoNT/E which target the same substrate, elicits a much shorter recovery.
  • SNAP-25A and SNAP-25j ⁇ have different turnover rates, as previously proposed (amongst other possibilities; Eleopra et al, 1998)? And if so why?
  • Various levels of SNAP-25A were detected at the original motor nerve terminals by immunocytochemistry from day 3 to 40 following injection of BoNT/A.
  • BoNT/E did initiate a series of reactions culminating in the removal of SNAP- 25 A most probably by endocytosis and retrograde transportation.
  • SNAP-25A can enter a SNARE complex, thereby rendering it exocytosis-incompetent and, furthermore, inaccessible to newly-synthesised SNAP-25.
  • BoNT/A treatment or SNAP-25A over-expression increases the number of docked vesicles in HIT-T15 insulinoma cells.
  • BoNT/E completely blocks Ca2+-activated exocytosis of large dense core vesicles whereas the BoNT/A-induced inhibition was only partial.
  • BoNT/E after BoNT/A drastically decreases this BoNT/A-insensitive exocytosis in permeabilised chromaffin cells.
  • SNAP-25 ⁇ is incapable of entering this SNARE complex, leaving it unprotected and exposed to clearance mechanisms operating within motor nerve terminals.
  • the constitutive pathway is known to be responsible for delivery of newly synthesised SNAP-25 through a cycle of exocytosis (Gonzalo et al, 1999; Kelly et al, 1993; O'SuUivan et al, 1999) and for replacement of defective SNAP-25 from the plasma membrane through vesicle- mediated recycling (Walch-Solimena et al, 1995).
  • Our results suggest that only SNARE-embedded SNAP-25 is involved in exo-endocytosis and that it is differentially turned-over with regard to other forms of SNAP-25. Under physiological conditions, SNAP-25pL is used by regulated exocytosis to operate in conjunction with the other SNAREs.
  • SNAP-25] ⁇ cannot assemble into the ternary SNARE complex, it is therefore expelled from the plasma membrane by constitutive endocytosis coupled with retrograde transport and replaced by newly-synthesised SNAP-25.
  • SNAP-25 A enters the ternary SNARE complex and is integrated into the regulated pathway competing with SNAP25pL for release sites. It is suggested that SNAP-25A could inhibit passage from the regulated exo-endocytotic pathway to retrieval of material by constitutive endocytosis and retrograde transport. Further experiments are needed to test this hypothesis which should be addressed with a more suitable model than NMJ in the hope of tackling the difficult question regarding the fate of the truncated products, and bringing clarity to such a complex series of events.
  • Botulinum neurotoxin-A selectively cleaves the synaptic protein SNAP-25. Nature 365, 160-163. Brown, M.C., Holland, R.L. and Hopkins, W.G. (1981) Motor nerve sprouting. Annl. Rev. Neurosci. 4, 17-42.
  • Botulinum neurotoxin Cl cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells - correlation with its blockade of catecholamine release. Biochem. 35, 2630-2636.
  • Botulinum-G neurotoxin cleaves vamp/synaptobrevin at a single Ala- Ala peptide-bond. J. Biol. Chem. 269, 20213-20216.
  • Botulinum neurotoxin serotype-F is a Zn ⁇ + -endopeptidase specific for vamp/synaptobrevin. J. Biol. Chem. 268, 11516-11519.

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Abstract

L'invention concerne un procédé pour traiter un patient victime d'une intoxication à la toxine botulique A (BoNT/A) ou à la toxine botulique C1 (BoNT/C1), consistant à administrer à ce patient de la toxine botulique E (BoNT/E) ou un polynucléotide codant et pouvant exprimer BoNT/E, ou un fragment dérivable par clivage du polypeptide associé à la vésicule synaptique de 25 kDa (SNAP-25) ou d'un variant de celui-ci par BoNT/E (SNAP-25E) ou un polynucléotide codant et pouvant exprimer SNAP-25E. Le patient peut être atteint de botulisme contracté naturellement ou accidentellement ou il peut avoir subi une injection de BoNT/A ou de BoNT/C1 à des fins médicales. L'invention concerne par ailleurs un procédé pour traiter un patient nécessitant une inhibition de courte durée de l'exocytose dans une de ses cellules, consistant à administrer à ce patient de la BoNT/E ou un polynucléotide codant et pouvant exprimer BoNT/E, ou un fragment dérivable par clivage du polypeptide associé à la vésicule synaptique de 25 kDa (SNAP-25) ou d'un variant de celui-ci par BoNT/E (SNAP-25E) ou un polynucléotide codant et pouvant exprimer SNAP-25E, sans administration de BoNT/A, B, C, F ou G. Le patient peut nécessiter une inhibition de l'exocytose d'une durée inférieure à 14 jours.
PCT/GB2002/002087 2001-05-04 2002-05-07 Bont/e ou snap-25e utilises pour traiter une intoxication a la toxine botulique a ou c1 et inhiber la contraction musculaire Ceased WO2002089834A1 (fr)

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US7172764B2 (en) 2003-11-17 2007-02-06 Allergan, Inc. Rescue agents for treating botulinum toxin intoxications
EP1644739B1 (fr) * 2003-07-04 2008-04-23 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procede de mise en evidence d un evenement moleculaire dans une cellule grace a des proteines marqueurs fluorescentes
WO2010009892A3 (fr) * 2008-07-24 2010-05-14 Bcn Peptides, S.A. Compositions destinées au traitement de la douleur et/ou de l'inflammation
EP2337790B1 (fr) * 2008-08-29 2016-07-13 Merz Pharma GmbH & Co. KGaA Neurotoxines clostridiennes ayant une persistence changée
KR101900582B1 (ko) 2013-12-23 2018-09-19 더블린 시티 유니버시티 만성 통증에 대한 다중프로테아제 치료법
CN110691579A (zh) * 2017-03-22 2020-01-14 邦蒂公司 用于治疗的肉毒杆菌神经毒素
WO2020065249A1 (fr) * 2018-09-28 2020-04-02 Ipsen Biopharm Limited Utilisations thérapeutiques et cosmétiques du sérotype e de la neurotoxine botulique
EP3600385A4 (fr) * 2017-03-22 2021-04-07 Bonti, Inc. Neurotoxines de botulinum pour le traitement de lésions traumatiques
RU2800604C2 (ru) * 2018-09-28 2023-07-25 Ипсен Биофарм Лимитед Терапевтическое и косметическое применение нейротоксина ботулина серотипа e
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EP1644739B1 (fr) * 2003-07-04 2008-04-23 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procede de mise en evidence d un evenement moleculaire dans une cellule grace a des proteines marqueurs fluorescentes
US7172764B2 (en) 2003-11-17 2007-02-06 Allergan, Inc. Rescue agents for treating botulinum toxin intoxications
WO2006011966A1 (fr) * 2004-06-30 2006-02-02 Allergan, Inc. Optimisation d'expression de toxine botulinum active de type e
US7825233B2 (en) 2004-06-30 2010-11-02 Allergan, Inc. Optimizing expression of active Botulinum Toxin type E
WO2010009892A3 (fr) * 2008-07-24 2010-05-14 Bcn Peptides, S.A. Compositions destinées au traitement de la douleur et/ou de l'inflammation
CN102164610A (zh) * 2008-07-24 2011-08-24 Bcn肽类股份有限公司 用于治疗疼痛和/或炎症的组合物
JP2011528678A (ja) * 2008-07-24 2011-11-24 ベーセーエネ ペプティデス，エセ．ア． 疼痛および／または炎症治療のための組成物
CN102164610B (zh) * 2008-07-24 2014-04-09 Bcn肽类股份有限公司 用于治疗疼痛和/或炎症的组合物
AU2009273471B2 (en) * 2008-07-24 2014-04-17 Bcn Peptides, S.A. Compositions for the treatment of pain and/or inflammation
RU2515054C2 (ru) * 2008-07-24 2014-05-10 БКН Пептидес, С.А. Композиции для лечения боли и/или воспаления
AU2009273471C1 (en) * 2008-07-24 2014-08-21 Bcn Peptides, S.A. Compositions for the treatment of pain and/or inflammation
EP2337790B1 (fr) * 2008-08-29 2016-07-13 Merz Pharma GmbH & Co. KGaA Neurotoxines clostridiennes ayant une persistence changée
KR101900582B1 (ko) 2013-12-23 2018-09-19 더블린 시티 유니버시티 만성 통증에 대한 다중프로테아제 치료법
CN110691579A (zh) * 2017-03-22 2020-01-14 邦蒂公司 用于治疗的肉毒杆菌神经毒素
EP3600221A4 (fr) * 2017-03-22 2021-01-13 Bonti, Inc. Neurotoxines botuliques pour utilisation en thérapie
EP3600385A4 (fr) * 2017-03-22 2021-04-07 Bonti, Inc. Neurotoxines de botulinum pour le traitement de lésions traumatiques
US11260114B2 (en) 2017-03-22 2022-03-01 Bonti, Inc. Botulinum neurotoxins for use in therapy
AU2018237198B2 (en) * 2017-03-22 2022-09-01 Bonti, Inc. Botulinum neurotoxins for use in therapy
WO2020065249A1 (fr) * 2018-09-28 2020-04-02 Ipsen Biopharm Limited Utilisations thérapeutiques et cosmétiques du sérotype e de la neurotoxine botulique
CN112739320A (zh) * 2018-09-28 2021-04-30 益普生生物制药有限公司 肉毒杆菌神经毒素血清型e的治疗和美容用途
JP2022502446A (ja) * 2018-09-28 2022-01-11 イプセン バイオファーム リミテッドIpsen Biopharm Limited ボツリヌス神経毒血清型ｅの治療的及び美容的使用
RU2800604C2 (ru) * 2018-09-28 2023-07-25 Ипсен Биофарм Лимитед Терапевтическое и косметическое применение нейротоксина ботулина серотипа e
JP2024010092A (ja) * 2018-09-28 2024-01-23 イプセン バイオファーム リミテッド ボツリヌス神経毒血清型ｅの治療的及び美容的使用
US20230405098A1 (en) * 2022-06-16 2023-12-21 Innomed Technologies, Inc. Type e botulinum toxin to treat botulism
WO2023245189A1 (fr) * 2022-06-16 2023-12-21 Innomed Technologies, Inc. Toxine botulique de type e pour traiter le botulisme

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