WO2021144794A1

WO2021144794A1 - Compounds for use in treatment and/or prevention of clostridial neurotoxins intoxication

Info

Publication number: WO2021144794A1
Application number: PCT/IL2021/050040
Authority: WO
Inventors: Alon BEN DAVID; Ada BARNEA; Ran ZICHEL
Original assignee: The Israel Institute of Biological Research (IIBR)
Priority date: 2020-01-13
Filing date: 2021-01-13
Publication date: 2021-07-22
Also published as: IL272003A

Abstract

The invention concerns compounds and methods for treating and/or preventing intoxication by clostridial neurotoxins.

Description

COMPOUNDS FOR USE IN TREATMENT AND/OR PREVENTION OF CUOSTRIDIAU NEUROTOXINS INTOXICATION

TECHNOUOGICAU FIEUD

The present disclosure relates to compounds that inhibit the binding of clostridial neurotoxins to their receptor, synaptic vesicle glycoprotein 2 (SV2) and to their therapeutic uses.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

[1] WO 2008/041966.

[2] WO 2011/022721.

[3] US 7,947,717.

[4] US 8,404,728.

[5] US 2018/147223.

[6] Duplantier et al., 2016. Curr Top Med Chem 16:2330-2349.

[7] Pirazzini and Rossetto, 2017. Expert Opin Drug Discov 12:497-510.

[8] Batistatou and Greene, 1991. J. Cell Biol. 115: 461-471.

[9] Rosenbaum et al., 1997. Vision Res. Vol. 37, No. 24: 3445-3451.

[10] Hashem et al., 2009. PloS One Vol. 4, issue 12: e8350.

[11] Rinne et al., 1975. J. Neurol. Vol. 211, issue 1: 1-9.

[12] Dong et al., 2006. Science 312:592-596.

[13] Diamant et al., 2014. PLoS One 9:e87089.

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter. BACKGROUND

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. The toxins are produced by the gram-positive spore-forming bacterium Clostridium botulinum. There are at least seven known serotypes of BoNTs designated A-G. Antibodies raised against one serotype can only neutralize the toxic effects of the BoNT serotype against which they were raised and not the effects of other BoNT serotypes.

All BoNT serotypes share a common architecture that consists of the following three domains responsible for the different steps in the intoxication mechanism: 1. The receptor binding domain (also known as the Hc-fragment); 2. The translocation domain; and 3. The catalytic domain. Following exposure to BoNTs, the neurotoxins bind specific receptors on motor neurons and disable the ability of the cells to transmit the neurotransmitter acetyl choline to muscle cells. This results in flaccid muscle paralysis and can lead to respiratory failure and eventually to death.

Currently, the only approved therapy for botulinum intoxication (botulism) is antitoxin, namely an antibody preparation, originating mostly from vaccinated horses, which have the capacity to neutralize the botulinum toxin in the bloodstream. Although effective, antitoxin therapy suffers from several disadvantages. First, the administration of a large dose of a foreign protein (namely an antibody) can cause severe side effects, including an anaphylactic shock. Due to secondary immunologic reaction against the equine (horse) antibodies, the antitoxin can only be administered for a single intoxication event per patient. Second, antitoxin therapy is very expensive due to the requirement for horses and restricted safety facilities dictated by working with the hazardous neurotoxin. Third, antibodies are thermolabile and require a so called “cold chain” delivery, which limits antitoxin distribution.

Due to at least the above reasons, there is a motivation for developing next generation therapy for botulism that will consist of small molecules that inhibit the neurotoxin. The production costs of small molecules are relatively low. The immune system does not react against small molecules, and therefore such therapy is safer than antitoxin therapy, and can be administered repeatedly and even for prophylactic purposes. Furthermore, small molecules are generally stable and do not require a cold chain delivery.

Small molecules can potentially target each one of the neurotoxin domains that are responsible for the different intoxication steps, i.e., receptor binding, toxin translocation, and enzymatic proteolysis of cytoplasmic SNARE (soluble N- ethylmaleimide- sensitive factor attachment protein receptor) proteins. Compounds that may potentially inhibit botulinum neurotoxins are disclosed for example in WO 2008/041966, WO 2011/022721, US 7,947,717, US 8,404,728, US 2018/147223 (1- 5). Efforts are being currently taken to find small molecule inhibitors for the catalytic domain of the toxin that will inactivate intracellular toxin (6, 7). The effect of small molecules on the catalytic domain can be relatively easily evaluated by measuring the enzymatic activity of the catalytic domain on synthetic substrates. Indeed, several small molecules were found to date to inhibit the catalytic domain of BoNTs, with dissociation constants in the nanomolar range (6, 7). However, only a limited therapeutic effect was reported thus far in vivo for catalytic domain inhibitors, due to their limited cell entry.

Aurintricarboxylic acid (also termed ATA) is a general endonuclease inhibitor that was shown to inhibit neuronal apoptosis (8), and to protect cells from ischemic cell damage (9). ATA was also shown to be an inhibitor of influenza A and B vims neuraminidases (10). Benserazide is an aromatic L-amino acid decarboxylase or DOPA decarboxylase inhibitor that was shown to be effective in the treatment of Parkinson's disease in combination with Levodopa (11). 6-hydroxy-DL-DOPA (6-OH-DL-DOPA) is a small molecule precursor of the catecholaminergic neurotoxin 6-hydroxydopamine. None of these molecules has been previously suggested as inhibitors of botulinum neurotoxins.

GENERAL DESCRIPTION

By a first one of its aspects the invention provides a compound having the structure (I):

wherein, - 4 - each of R₁, R₂ and R₃, independently of the other, is selected from -H, -C(=0)-OH, -OH, C₁-C₅alkylene and C₂-C₅alkenylene, and wherein one of said R₁, R₂ and R₃ is ortho to the -OH group; or a salt thereof for use in treating and/or preventing intoxication by at least one clostridial neurotoxin.

In some embodiments the compound for use according to the invention is wherein each of R₁ and R₂ is -OH.

In other embodiments the compound for use according to the invention has the structure (II):

wherein R₃ is selected from -C(=0)-OH, -OH and C₁-Csalkylene.

In further embodiments the compound for use according to the invention is wherein C₁-Csalkylene is substituted.

In still further embodiments the compound for use according to the invention is wherein the C₁-Csalkylene is substituted by a group selected from -OH, -NR’R ’R ” and -C(=0)-OR₄, wherein each of R’ and R” is independently selected from -H, C₁-Csalkyl and C₂-C5alkenyl, wherein R”’ is absent or is selected from -H, C₁-Csalkyl and C₂- Csalkenyl, and wherein R₄ is -H, C₁-Csalkyl or C₂-C5 alkenyl. In certain embodiments the compound for use according to the invention is wherein -NR’R ’R ” is selected from -NH-, -NH2, -NHR’ and -NH2R’”, and wherein each of R’ and R’” is as defined herein above.

In some embodiments the compound for use according to the invention is wherein C₁-Csalkylene is a group having the structure (III):

wherein n is 0 or 1, each of R₅’ and Rs, independently of the other, is absent or is selected from -H, - OH, -NR’R”R”’ and -C(=0)-OR₄,

R₄ is -H, C₁-Csalkyl or C₂-C₅alkenyl,

X is -C(=0)- and -NH-NH-C(=0)-R₆-,

R₆ is an optionally substituted C₁-Csalkylene, wherein substitution is by a group selected from -OH, -NR’R”R”\ -C(=0)-OR₄, each of R’ and R” is independently selected from -H, C₁-C₅alkyl and C₂- Csalkenyl, and wherein

R”’ is absent or is selected from -H, C₁-Csalkyl and C₂-C5alkenyl.

In specific embodiment the compound for use according to the invention is wherein n is 1. In other specific embodiments the compound for use according to the invention is wherein n is zero.

In certain embodiments the compound for use according to the invention is wherein R₅’ is absent (namely the position substituted by R₅’ is H) and R₅ is selected as defined herein.

In other embodiments the compound for use according to the invention is wherein Rs is -NR’R”R”’, and wherein each of R’, R” and R”’ is as defined herein.

In further embodiments the compound for use according to the invention is wherein n is 1, R₅’ is -H, R₅ is NH₂ and X is -C(=0).

In certain specific embodiments the compound for use according to the invention is having the structure (1):

In further specific embodiments the compound for use according to the invention is wherein X is -NH-NH-C(=0)-R₆- and R₆ is optionally a substituted C₂alkylcnc. In additional embodiments the compound for use according to the invention is wherein the C₂alkylene is substituted by -NR’R”R”’, and wherein each of R’, R” and R’” is as defined herein.

In some embodiments the compound for use according to the invention is having the structure (2):

In other embodiments the compound for use according to the invention is wherein one or R₁, R₂ and R₃ is -H.

In further embodiments the compound for use according to the invention is wherein one of R₁, R₂ and R₃ is -C(=0)-OH.

In still further embodiments the compound for use according to the invention is wherein one of R₁, R₂ and R₃ is a C₂- C₅alkylene being optionally substituted.

In certain embodiments the compound for use according to the invention is wherein one of R₁, R₂ and R₃ is a C₂-C₅alkenylene being optionally substituted.

In further embodiments the compound for use according to the invention is wherein the C₂-C₅alkenylene group is of structure (IV):

wherein

R7 and Rs together with the carbon atom to which they are bonded to form a 6- memebred ring, and wherein

R₉ is an optionally substituted aryl.

In certain embodiments the compound for use according to the invention is wherein R₇ and R₈ together with the carbon atom to which they are bonded form a ring structure having the structure (V):

wherein R₁o is selected from -C(=0)-0R_a and -C(=0)-R_b, wherein each of R_a and R_b is independently selected from -H, -OH, C₁-Csalkyl, C₁-Cshydroxyalkyl; and wherein

R9 is an optionally substituted aryl.

In specific embodiments the compound for use according to the invention is wherein R₁o is -C(=0)-0R_a, and R_a is -H; wherein, R9 is an optionally substituted aryl; and wherein structure (V) is of structure (VI):

In other embodiments the compound for use according to the invention is wherein R9 is a substituted aryl, of the structure (VII):

wherein each of R₇ and R₈ are as defined herein above, each of Rn and R12, independently of the other is selected from -H, -OH, -C(=0)- OR_f, -C(=0)-R_g and wherein each of R_f and R_g is independently selected from -H, C₁-C₅alkyl and C₁- C₅hydroxyalkyl. In some embodiments the compound for use according to the invention is wherein R11 is -OH and R₁₂ is -C(=0)-OH; the group having the structure (VII) is of the structure

(VIII):

In some specific embodiments the compound for use according to the invention is having the structure (3):

In certain embodiments the compound for use according to the invention is wherein said clostridial neurotoxin is a Botulinum neurotoxin or a tetanus neurotoxin.

In some embodiment the compound for use according to the invention is wherein said intoxication is botulism or tetanus intoxication.

The invention in a further aspect thereof provides a compound selected from compounds herein designated compound (1), compound (2) and compound (3) for use in treating and/or preventing botulism or tetanus intoxication. Still further, the invention provides an inhibitor of a clostridial neurotoxin, wherein the inhibitor is any one of the compounds according to the invention.

In some embodiments the inhibitor according to the invention is wherein said clostridial neurotoxin is a Botulinum neurotoxin, a tetanus neurotoxin or a combination thereof.

In other embodiments the inhibitor according to the invention is wherein said Botulinum neurotoxin is Botulinum neurotoxin A (BoNT/A), Botulinum neurotoxin E (BoNT/E), Botulinum neurotoxin D (BoNT/D) or Botulinum neurotoxin F (BoNT/F) or any combination thereof.

By another one of its aspects the invention provides a method for preventing and/or treating intoxication by a clostridial neurotoxin in a subject in need thereof, comprising administering to said subject an effective amount of at least one compound according to the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In some embodiments the method according to the invention is wherein said intoxication by a clostridial neurotoxin is caused by at least one of BoNT/A, BoNT/E, BoNT/D, BoNT/F and TeNT.

In other embodiments the method according to the invention is wherein said intoxication by a clostridial neurotoxin is botulism.

In further embodiments the method according to the invention is wherein said intoxication is botulism caused by at least one of BoNT/A or BoNT/E.

By a further aspect thereof the invention provides a method for inhibiting the binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2) in a subject, said method comprising administering to said subject an effective amount of at least one compound according to the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof. In some embodiments the method according to the invention is wherein the clostridial neurotoxin is at least one of BoNT/A, BoNT/E, BoNT/D, BoNT/F, and tetanus toxin.

In other embodiments, the method according to the invention is wherein said subject has been exposed to Clostridium bacteria, to a Botulinum neurotoxin or to a tetanus neuro toxin. In further embodiments the method according to the invention is wherein said subject is at risk of exposure to Clostridium bacteria , to a Botulinum neurotoxin or to a tetanus neuro toxin.

In certain embodiments the method according to the invention is wherein said subject is a human.

In specific embodiments the method according to the invention is wherein said intoxication by a clostridial neurotoxin is foodborne botulism, infant botulism, wound botulism or inhalation botulism.

In some embodiments the method according to the invention is wherein said at least one compound according to the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered to said subject as a single dosage unit or as multiple dosage units.

In other embodiments the method according to the invention is wherein said at least one compound according to the invention or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered by intravenous, intramuscular, parenteral, oral, nasal, or rectal administration.

In certain embodiments the method according to the invention wherein said method further comprises administration of at least one additional therapeutic agent.

In specific embodiments the method according to the invention is wherein said of at least one additional therapeutic agent is at least one antibody directed against a clostridial neuro toxin.

By a further aspect thereof the invention provides a pharmaceutical composition comprising at least one compound according to the invention for treating or preventing intoxication by at least one clostridial neurotoxin.

BRIEF DESCRIPTION OF THE DRAWINGS

For better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. lA-Fig. 1C: Dose-response curve for inhibition of β-gal-Hc/A - GST-SV2C interaction by Aurintricarboxylic acid (Fig. 1A); Benserazide (Fig IB); and 6-hydroxy - DL-DOPA (Fig. 1C). X axis - compounds concentration (mM); Y axis - Fluorescence intensity (excitation - 360 nm; emission - 460 nm). The dotted line indicates the IC50 value on the exponential X axis.

Fig. 2A-Fig.2C: Survival curves of mice exposed to four LD50 of BoNT/A and treated with Aurintricarboxylic acid (ATA 3.125mg, Fig. 2A); Benserazide (benz 12.5mg, Fig 2B); or 6-hydroxy-DL-DOPA (6-OH-DL-DOPA 2.5mg, Fig. 2C). The graphs show percent survival over time (hours).

Figure 3: A bar graph showing antibody detection (indicated as absorbance at 405nm) of receptor (SVC2)-bound Hc/E fragment in the absence (control) and in the presence of Aaurintricarboxylic acid.

Fig. 4A-Fig.4C: Survival curves of mice exposed to 4 LD50 of BoNT/E and treated with Benserazide (benz, Fig. 4A); 6-hydroxy-DL-DOPA (6-OHD, Fig. 4B); Aurintricarboxylic acid (ata, Fig. 4C) or phosphate buffered saline (pbs). The graphs show percent survival over time (hours).

DETAILED DESCRIPTION OF EMBODIMENTS

The present disclosure describes the novel use of a family of compounds as inhibitors of clostridial neurotoxins. The compounds of the invention conferred beneficial therapeutic effects in mice exposed to 4 LD50 of botulinum neurotoxin A (BoNT/A). The compounds disclosed herein have been discovered by screening of a compound library using a novel assay for screening of inhibitors for the binding of BoNT/A receptor binding domain to its receptor.

As detailed below, based on a high throughput (HTS) assay developed by the inventors for screening of small molecule binding inhibitors (SMBIs) of clostridial neurotoxins to their receptors, various SMBIs were identified and validated. The assay was originally used to select effective anti BoNT/A SMBIs, and indeed the selected SMBIs protected mice from a lethal dose of BoNT/A. Moreover, the selected SMBIs were found to be potent also against BoNT/E (namely the compounds showed multivalent potency). The SMBIs of the invention may present an advantage over traditional antitoxin in terms of cost of production, temperature- stability, safety and multi- valent potency and have the potential to expand the therapeutic arsenal for clostridial neurotoxin intoxication both in the form of terror events or as an advanced medical response for remote field forces. In one aspect, the invention thus provides an inhibitor of at least one clostridial neurotoxin, the inhibitor having the structure (I):

wherein each of R₁, R₂ and R₃, independently of the other, is selected from -H, -C(=0)- OH, -OH, C₁-Csalkylene and C₂-C₅alkenylene, provided that one of said R₁, R₂ and R₃ is ortho to the -OH group, and provided that none of said R₁, R₂ and R₃ is an optionally substituted pyran.

In some embodiments, the C₁-Csalkylene is C₁-C₃alkylene or C₂-C₃alkylene.

In some embodiments, the C₂-C₅alkenylene is C₂-C₃alkenylene.

In some embodiments, in a compound of structure (I), each of R₁ and R₂ is -OH. In some embodiments, the inhibitor of structure (I) has the structure (II):

wherein R₃ is selected from -C(=0)-OH, -OH and C₁-Csalkylene.

In a compound of structure (I), each of groups R₁, R₂ and R₃, irrespective of its selection may be positioned on any of the ring carbon atoms relative to the phenolic (- OH) group shown. Thus, groups R₁, R₂ and R₃ may be positioned ortho-, meta- or para- to the phenolic (-OH) group. In some embodiments, R₁, R₂ and R₃ are positioned on neighboring carbon atoms relative to the phenolic group. Where the ring carbon atom bearing the phenolic (-OH) group may be designated carbon 1, R₁, R₂ and R₃ may be positioned at positions 2, 3 and 4; 2, 3 and 5; 2, 3 and 6; 2, 4 and 5; 2, 4 and 6; 3, 4 and 5; and so on.

In a similar fashion, in a compound of structure (II), each of the two variable -OH groups (being R₁ and R₂) may be positioned at any position along the ring relative to the phenolic (-OH) group or relative to the R₃ group or relative to each other. In some embodiments, the three -OH groups in a structure (II), i.e., including the phenolic (-OH) group and the two variable -OH groups, are positioned on neighboring carbon atoms or may be separated by -H or by group R₃.

In some embodiments, in a compound having structure (I) or (II), wherein the C₁- Csalkylene is substituted. The substitution may be by a group selected from -OH, - NR’R”R”’ and -C(=0)-0R₄, wherein each of R’ and R” is independently selected from -H, C₁-Csalkyl and C₂-Csalkenyl, wherein R”’ is absent or is selected from -H, C₁-Csalkyl and C₂-C5alkenyl, and wherein R4 is -H, C₁-Csalkyl or C₂-C5 alkenyl.

In some embodiments, -NR’R”R”’ is selected from -NH-, -NH2, -NHR’ and - NH2R’”, wherein each of R’ and R’” is as defined herein.

In some embodiments, the C₁-Csalkylene is a group having the structure (III):

wherein n is 0 or 1, each of R₅’ and R₅, independently of the other, is absent or is selected from -H, -

OH, -NR’R”R”’ and -C(=0)-0R₄,

R4 is -H, C₁-Csalkyl or C₂-C₅alkenyl,

X is -C(=0)- and -NH-NH-C(=0)-R₆-,

R₆ is an optionally substituted C₁-Csalkylene, wherein substitution is by a group selected from -OH, -NR’R”R”\ -C(=0)-OR₄, each of R’ and R” is independently selected from -H, C₁-Csalkyl and C₂- Csalkenyl, and wherein

R”’ is absent or is selected from -H, C₁-Csalkyl and Ci-Csalkcnyl.

In some embodiments, in the group having the structure (III), n is 1 or n is zero.

In some embodiments, in the group having the structure (III), Rs’ is absent (namely the position substituted by R₅’ is H) and R₅ is selected as defined herein.

In some embodiments, in the group having the structure (III), R₅ is -NR’R”R”’, wherein each of R’, R” and R”’ is as defined herein.

In some embodiments, in the group having the structure (III), n is 1, R₅’ is -H, R₅ is NH₂ and X is -C(=0).

In some embodiments, a compound according to structure (I) or structure (II) is a compound having the structure (1):

In some embodiments, in the group having the structure (III), X is -NH-NH- C(=0)-R₆- and R₆ is optionally a substituted C2alkylene.

In some embodiments, the Cialkylcnc is substituted by -NR’R”R”’, wherein each of R’, R” and R’” is as defined herein.

In some embodiments, a compound according to structure (I) or structure (II) is a compound having the structure (2): R₅

_{In some embodiments, in a compound according to structure (I) or structure (II)} _{one of R1, R2 and R3 is -H. In some embodiments, one of R1, R2 and R3 is -C(=0)-0H.} _{In some embodiments, one of R1, R2 and R3 is a C2-Csalkenylene being optionally} _{substituted. In some embodiments, one of R1, R2 and R3 is a C2-C5alkenylene being} _{optionally substituted.} _{In some embodiments, the C2-C5alkenylene group is of structure (IV):}

w_herein _{R7 and R8 together with the carbon atom to which they are bonded to form a 6-} _{memebred ring, and wherein} _{R9 is an optionally substituted aryl.} _{As used herein, groups R7 and Rs together with the carbon atom to which they are} _{bonded form a ring structure that comprises 6 atoms. The 6-memebered ring structure} _{may be substituted and/or comprise one or more endo-cyclic or exo-cyclic double bonds.} _{The atoms making up the 6-memebred ring structure are typically carbon atoms. In some} _{embodiments, the 6-memebred ring structure is a 6-memebred carbocyclic ring structure.} _{In other embodiments, the 6-memebred ring structure comprises one or more heteroatoms} _{selected from N, O and S.} _{In some embodiments, the 6-memebred ring structure is a carbocyclic that is} _{optionally substituted. In some embodiments, the 6-memebred ring structure is of the} _{structure (V):}

_{Ri o} _(V) _wherein Rio is selected from -C(=0)-0R_a and -C(=0)-R_b, wherein each of R_a and R_b is independently selected from -H, -OH, C₁-Csalkyl, C₁-C₅hydroxyalkyl; and wherein R9 is an optionally substituted aryl.

In some embodiments, Rio is -C(=0)-0R_a, and R_a is -H.

In some embodiments, R9 is an optionally substituted aryl.

In some embodiments, the ring structure (V) is of the structure (VI):

wherein each of R7 and R₈ are as defined herein, each of Rn and R₁₂, independently of the other is selected from -H, -OH, -C(=0)- OR_f, -C(=0)-R_g and wherein each of R_f and R_g is independently selected from -H, C₁-Csalkyl and C₁- C₅hydroxyalkyl.

In some embodiments, Rn is -OH and R₁₂ is -C(=0)-OH.

In some embodiments, the group of structure (VII) is of the structure (VIII):

wherein R₇ and R₈ are as defined herein.

In some embodiments, a compound of structure (I) or (II) is a compound having the structure (3):

The invention further provides a compound selected from compounds herein designated compound (1), compound (2) and compound (3).

The group ^"C1-C5alkyl" is an aliphatic hydrocarbon group comprising between 1 and 5 carbon atoms arranged in a linear or branched form. As indicated herein, the alkyl may be substituted or unsubstituted, as defined. Non-limiting examples of Cl-C5alkyl include methyl, ethyl, propyl, butyl, pentyl, iso-propyl, tert-butyl, iso-butyl and others. The “ C₂-C₅alkenyl ” comprises between 2 and 5 carbon atoms, and between 1 and 3 double bonds.

As used herein, the group “ C₂-C₅alkylene’ ’ is a divalent aliphatic hydrocarbon group comprising between 1 and 5 carbon atoms arranged in a linear or branched form. The alkylene moiety may be substituted by a group selected from -OH, -NR’R ’R ” and -C(=0)-0R₄, as defined herein.

The term “ C₂-C₅alkenylene ” refers to a carbon chain comprising between 2 and 5 carbon atoms and between 1 and 3 double bonds.

The group -NR’R”R”’ designates an amine group or an ammonium group which may be selected from -NH-, -NH2, -NHR’ and -NH2R’”, wherein each of R’ and R’” is as defined herein. The amine may be a primary amine, a secondary amine, a tertiary amine or a quaternary amine. Where the amine is a quaternary it comprises a counter ion (an anion) which may be selected amongst organic and inorganic anions.

Preparation of the compounds as herein defined is known to a person of skill in the art.

The invention further provides use of a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3) in the preparation of a pharmaceutical composition for treating and/or preventing intoxication by at least one clostridial neurotoxin and/or ameliorating at least one of the symptoms associated therewith.

The invention further provides use of a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3) in the preparation of a pharmaceutical composition for inhibiting clostridial neurotoxin binding to its receptor Synaptic vesicle glycoprotein 2 (SV2).

The protein receptor for several of the clostridial neurotoxins is synaptic vesicle glycoprotein 2. The term “receptor synaptic vesicle glycoprotein 2”, also referred to herein as “SV2” and as known in the art refers to a family of transporter-like proteins that are located in synaptic neurotransmitter-containing vesicles. The three SV2 genes in mammals encode three isoforms, namely SV2A, SV2B and SV2C.

The SV2s are membrane proteins, found in synaptic and endocrine secretory vesicles of vertebrates. These proteins have 12 putative transmembrane domains, and five loops that are directed towards the vesicle lumen. Dong et. al. (12) demonstrated that Botulinum neurotoxin A (BoNT/A) binds the fourth luminal loop of the synaptic vesicle glycoprotein 2C (SV2C) with the highest affinity.

Similarly, Botulinum neurotoxin E (BoNT/E) also uses the fourth luminal loop of SV2 as a receptor. The isoforms SV2A and SV2B are the preferred target for BoNT/E. Additionally, it is believed that SV2s are the protein receptor for Botulinum neuro toxin D (BoNT/D), Botulinum neurotoxin F (BoNT/F) and tetanus toxin (TeNT).

The present invention thus provides inter alia a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3), for use in inhibiting binding of a clostridial neurotoxin to at least one of the SV2 receptor types, namely SV2A, SV2B or SV2C.

Still further the invention provides use of a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3), in the preparation of a pharmaceutical composition for inhibiting at least one of Botulinum neurotoxin A, E, D, F and tetanus toxin binding to any one of the receptors SV2A, SV2B or SV2C.

The invention further provides a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3), for use in treating and/or preventing intoxication by at least one clostridial neurotoxin and/or ameliorating at least one of the symptoms associated therewith.

The invention further provides a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3), for use in inhibiting clostridial neuro toxin binding to its receptor SV2.

Still further the invention provides a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3), for use in inhibiting at least one of Botulinum neurotoxin A, E, D, F or tetanus neurotoxin binding to its receptor SV2. In a specific embodiment the invention provides a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3), for use in inhibiting Botulinum neuro toxin A binding to its receptor SV2C.

Based on the assays performed and described herein, the following three compounds presented a high inhibition of the binding between the He fragment of BoNT/A and its receptor SV2C: 6-hydroxy-DL-dopa (herein designated compound (1)), Benserazide (herein designated compound (2)) Aurintricarboxylic acid (herein designated compound (3)).

Therefore the invention further provides a compound herein designated compound (1), (2) or (3) as described above for use in inhibiting at least one clostridial neuro toxin (for example Botulinum neuro toxin A) binding to its receptor SV2 (for example SV2C).

The term “inhibit”, “inhibiting” or "inhibition", as used herein, means the restriction, retardation, reduction, decrease or diminishing of intoxication by at least one clostridial neurotoxin (e.g. botulism or tetanus intoxication), or any symptom or phenotype associated therewith by at least about 1%-100%, about 5%-95%, about 10%- 90%, about 15%-85%, about 20%-80%, about 25%-75%, about 30%-70%, about 35%- 65%, about 40%-60% or about 45%-55%. Said restriction, retardation, reduction, decrease or diminishing of intoxication by at least one clostridial neurotoxin, or any symptom or phenotype associated therewith may also be by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,

35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,

65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99% or about 100%.

Examination of inhibition of clostridial neurotoxin binding to its receptor SV2 in the presence of the compound according to the present invention may be performed by any method known in the art, for example by the methods detailed in the Examples below.

By the term “ intoxication by at least one clostridial neurotoxin ” as used herein it is meant to refer to any disease caused by exposure of a subject to a clostridial neuro toxin. Clostridia, a genus of Gram-positive bacteria, includes several human pathogens, including the causative agents of botulism and tetanus. Clostridium species inhabit soils and the intestinal tract of animals, including humans. Clostridium include around 100 species, inter alia, Clostridium botulinum which can produce botulinum toxin in food or wounds and can cause botulism; Clostridium perfringens causes a wide range of symptoms, from food poisoning to cellulitis, fasciitis, and gas gangrene; Clostridium tetani which can produce tetanus neurotoxin and causes tetanus or the exotoxin tetanolysin, which is a hemolysin that causes destruction of tissues; and Clostridium sordellii that can cause a fatal infection in exceptionally rare cases after medical abortions.

The present invention relates to an intoxication caused by any clostridial neurotoxin. In certain embodiments the present invention relates to a clostridial neurotoxin which is a botulinum neurotoxin, a tetanus neurotoxin or a combination thereof.

Botulinum neurotoxins (BoNTs), as known in the art and as used herein, are bacterial proteins that cause the life-threatening disease botulism and are designated by the Centers for Disease Control and Prevention (CDC) “category A” agents. Eight antigenically distinct serotypes (designated A to H) are produced by several anaerobic species: Clostridium botulinum, Clostridium butyricum, Clostridium baratii and Clostridium argentinense. BoNTs A, B, E, and rarely, F serotypes are primarily related to human illness. The neurotoxin is a di-chain polypeptide consisting of a 100-kDa heavy chain (HC) joined by a disulphide bond to a 50-kDa light chain (LC). All BoNT serotypes exert similar mechanisms of action on their target nerve cells: initial binding of the C- terminal portion of the HC through ganglioside and protein receptors on the presynaptic cell surface, followed by internalization and translocation within the nerve ending, mediated by the N-terminal portion of the HC. Inside the nerve terminus, the neurotoxin LC, cleaves the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), which involved in the fusion and release of acetylcholine. As a result, acetylcholine transmission across neuromuscular junctions is blocked and symmetric descending flaccid paralysis occurs.

In some specific embodiments the present invention relates to botulinum neurotoxin produced by Clostridium botulinum. In one embodiment the present invention relates to botulinum neurotoxins that bind the receptor SV2. In certain specific embodiments the botulinum neurotoxin according to the present invention is Botulinum neurotoxin A (BoNT/A), Botulinum neurotoxin E (BoNT/E), Botulinum neurotoxin D (BoNT/D) or Botulinum neurotoxin F (BoNT/F) or any combination thereof.

In certain embodiments the intoxication according to the present invention is botulism or tetanus intoxication.

The terms ''botulism” and "botulinum intoxication" are used interchangeably herein and refer to a disease caused by exposure of a subject to a toxin ("botulinum toxin" also referred to herein as "botulinum neurotoxin") produced mainly by the bacterium Clostridium botulinum. As used herein the term encompasses all types of botulism including, but not limited to, foodbome botulism, infant botulism, inhalation botulism, and wound botulism. Diagnosis of botulinum intoxication is within the skills of a physician.

Following exposure of a subject to a "botulinum toxin", the botulinum toxin binds specific receptors on motor neurons and disable the ability of the cells to transmit the neurotransmitter acetyl choline to muscle cells. There are eight types of botulinum toxin, named type A-H. Botulinum neurotoxin A binds to the receptor SV2C.

Interestingly, as shown in the examples below, the three compounds herein designated compound (1), (2) or (3) as described above exhibited a significant therapeutic effect, suggesting that the compounds 6-hydroxy-DF-DOPA (6-OH-DF-DOPA, herein designated compound (1)), Benserazide (herein designated compound (2)) and Aurintricarboxylic acid (herein designated compound (3)) are inhibitors of BoNT/A, specifically the receptor binding domain thereof, and as such they appear to inhibit the attachment of the toxin to its cellular receptor. Furthermore, the above three compounds were also shown to inhibit BoNT/E in vivo, as demonstrated in the examples below.

Therefore the invention further provides a compound selected from compounds herein designated compound (1), compound (2) and compound (3) for use in treating and/or preventing botulism.

Tetanus neurotoxin (“tetanus toxin”, “spasmogenic toxin”, or “TeNT”) is an extremely potent neurotoxin produced by Clostridium tetani, in anaerobic conditions, causing the disease tetanus. Tetanus toxin is initially bound at the presynaptic terminals of the neuromuscular junction, then it is transported by motor neurons to the spinal cord. In the spinal cord, tetanus toxin is then transferred to inhibitory presynaptic terminals surrounding those motor neurons. The toxin then cleaves a vesicular synaptic membrane protein (VAMP, or synaptobrevin), resulting in inactivation of inhibitory neurotransmission that normally suppresses motor neuron and muscle activity. This action results in enhanced excitability and activation of the affected motor neurons.

The term “tetanus” used interchangeably with tetanus intoxication as known in the art is a bacterial infection characterized by muscle spasm. A widespread intoxication of tetanus toxin through the systemic circulation results in continuous involuntary muscle contraction, whereas local internalization and transport of the toxin results in a localized state of muscle hyperexcitability.

In specific embodiments the present invention provides a compound as herein defined or a pharmaceutical composition comprising thereof for use in treating and/or preventing tetanus intoxication. In further embodiments the present invention provides a compound selected from compounds herein designated compound (1), compound (2) and compound (3) for use in treating and/or preventing tetanus intoxication.

Still further the invention provides a pharmaceutical composition comprising at least one compound as herein defined, for example a compound selected from compounds herein designated compound (1), compound (2) and compound (3). In particular embodiments the pharmaceutical composition according to the present invention is for treating or preventing intoxication by at least one clostridial neurotoxin.

The invention further provides a pharmaceutical composition for treating and/or preventing botulism or tetanus intoxication and/or ameliorating at least one of the symptoms associated therewith comprising at least one compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3). The invention further provides a pharmaceutical composition for inhibiting clostridial neurotoxin binding to its receptor SV2 comprising a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3). Still further the invention provides a pharmaceutical composition for inhibiting botulinum neurotoxin A binding to its receptor SV2C comprising a compound of structure (I) or (II) as described above, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3). In some embodiments, the composition comprises a pharmaceutically acceptable carrier, excipient or diluent. The compound according to the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered by any route of administration. In some embodiments administration is by intravenous, intramuscular, parenteral, oral, nasal or rectal administration.

The pharmaceutically acceptable carrier, excipient or diluent is chemically inert to the active compounds, namely the compound of structure (I) or (II) as defined herein, for example a compound selected from the compounds herein designated compound (1), compound (2) and compound (3), and is selected to have no detrimental side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the particular compound used, as well as by the particular method used to administer a composition comprising the compound. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The formulations may be adapted for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, rectal, and vaginal administrations.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and com starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active compounds in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art. Compounds used in accordance with the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2, 2-dimethyl- l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxy- ethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-P-aminopriopionates, and 2-alkyl- imidazoline quaternary ammonium salts, and (3) mixtures thereof.

The parenteral formulations may contain from about 0.5 to about 25% by weight of the active compound in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

Compounds disclosed herein may be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4^th ed., pages 622-630 (1986).

The invention further provides a method of treatment and/or prevention and/or amelioration of intoxication by a clostridial neurotoxin and/or at least one of the symptoms of intoxication by a clostridial neurotoxin in a subject.

As used herein, the term “ treatment or prevention ” refers to the administering of a therapeutic amount of a compound as disclosed herein or a pharmaceutical composition of the present invention which is effective to ameliorate undesired symptoms associated with exposure to a clostridial neurotoxin, for example undesired symptoms associated with exposure to Botulinum neurotoxin A, to prevent the manifestation of such symptoms before they occur, to improve survival rate or more rapid recovery.

As used herein, the term "subject” refers to any animal that may be affected by botulism, including but not limited to cattle, sheep, and horses. In a specific embodiment the term subject refers to a human subject. By the term “subject” it is referred to a subject that has been exposed or to a subject at risk of exposure to Clostridium bacteria, a Botulinum neuro toxin or a tetanus neuro toxin.

In accordance with the method of the invention, said at least one compound of structure (I) or (II) as described above is administered about 24 hours before potential exposure to botulinum toxin.

In accordance with the method of the invention, said at least one compound of structure (I) or (II) as described above is administered about 48 hours after exposure to botulinum toxin.

In some embodiments, the method according to the present invention further comprises administration of at least one additional therapeutic agent.

By the term “ additional therapeutic agent ” as used herein it is meant to include any additional therapeutic agent for the treatment of intoxication by at least one clostridial neurotoxin (for example but not limited to Botulinum or tetanus intoxication) known to a skilled physician. The additional therapeutic agent may for example be a botulinum antitoxin, namely an antibody (or antibody fragment) preparation directed against the botulinum toxin, e.g. the FDA approved heptavalent botulinum antitoxin (HBAT).

In specific embodiments the at least one additional therapeutic agent as herein defined is at least one antibody directed against a Botulinum neurotoxin or to a tetanus neuro toxin.

The term "about” as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.

It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. The term “at least one ” is meant to indicate one, two, three, four, five, six, seven, eight, nine, ten or more.

Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present disclosure to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Experimental procedures b -gal- He/ A chimeric protein design and purification

A chimeric protein construct was prepared as described below, consisting of two proteins (polypeptides): (1) beta-galactosidase from Escherichia coli', and (2) the receptor binding domain of BoNT/A, also designated BoNT/A or the H_c fragment. The chimeric protein construct is referred to herein as “β-gal-Hc/A”. The sequence of the beta- galactosidase protein originating from E. coli BL21 is publically available, for example as detailed in gene bank code CAQ30819 and is denoted herein by SEQ ID NO. 1. The sequence of the He fragment protein of BoNT/A originating from Clostridium botulinum strain 62A is publically available, for example as detailed in gene bank code M30196, amino acids 872-1296 and is denoted herein by SEQ ID NO. 2. Beta-gal was set (i.e. positioned) on the N-terminus of the chimeric protein construct and the Hc-fragment on its C-terminus. The two proteins (polypeptides) were connected by a flexible linker with the sequence (GGGGS)3, denoted herein by SEQ ID NO. 3, and a Hisx6 tag (denoted herein by SEQ ID NO. 4) was added to the C-terminus of the chimeric protein construct (namely C-terminal to the Hc-fragment) in order to facilitate its purification using a nickel (Ni) column. The amino acid sequence of the chimeric protein construct prepared as described above is denoted by SEQ ID NO. 5.

Genes of clostridial origin are AT rich and their codon usage is different from that of E. coli. Therefore, a synthetic gene with optimized codon usage for protein expression in E. coli was prepared (GenScript). The gene was cloned into the expression vector pET- 9a to obtain the plasmid pET-9a-β-gal-Hc/A. The nucleic acid sequence encoding the chimeric protein construct prepared as described above, which was inserted into the plasmid pET-9a-B-gal-Hc/A, is denoted herein by SEQ ID NO. 6.

The chimeric protein construct was purified as detailed below. The plasmid pET- 9a-B-gal-Hc/A was transformed into E. coli BL21(DE3). A starter culture was prepared by inoculating a colony into 40 ml of TB media (comprising 24 g/1 yeast extract; 12 g/1 tryptone; 4 g/1 glycerol; 89 mM potassium phosphate) with kanamycin (30 pg/ml) in an Ultra Yield 250 ml (Thomson Instrument Company) shake flask, and the flasks were incubated at 37°C, 250 rpm. After 8 hours the starter culture was used to inoculate 1 liter of TB media with kanamycin, in two Ultra Yield 2.5 liter shake flasks, and the flasks were incubated at 18°C, 250 rpm for 40 hours. The culture was harvested by centrifugation.

The cell pellet was re-suspended in 100 ml binding buffer (20 mM imidazole; 20 mM sodium phosphate; 0.5 M NaCl; pH 7.4) and the cells were disrupted by sonication (Cole Parmer ultrasonic processor; 60% amplitude, 15 minutes). The cell extract was clarified by centrifugation (14,000 g, 30 min) and loaded onto a HisTrap FF 1 ml column (GE Healthcare) mounted on an AKTA Explorer FPLC system (GE Healthcare). The column was washed with 10 column volumes (CV) of binding buffer and 10 CV of binding buffer containing 40 mM imidazole. The protein was eluted from the column with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM Imidazole, pH 7.4). The purified protein was dialyzed against 50 mM sodium citrate buffer, pH 5.5, and stored at -70° C).

Expression and purification of a GST-SV2C construct

The chimeric receptor polypeptide is also referred to herein as “GST-SV2C”.

The protein receptor for BoNT/A is synaptic vesicle glycoprotein 2C (SV2C). Specifically, the toxin binds the fourth luminal loop of SV2C (12). The gene encoding the fourth luminal loop of sv2c (having a nucleic acid sequence denoted herein as SEQ ID NO. 7) was fused to S. japonicum glutathione-s-transferase (gst) gene (having a nucleic acid sequence denoted herein as SEQ ID NO. 8) to facilitate purification and enhance its solubility. The protein sequence of GST was taken from pET-41a vector and has a sequence denoted herein as SEQ ID NO. 9. The protein sequence for the fourth luminal loop of SV2C was taken from Mus musculus and has a sequence denoted herein as SEQ ID NO. 10. The fusion receptor protein consists of GST on its N-terminal and the fourth luminal loop of SV2C on its C-terminal (GST-SV2C). A synthetic gene with optimized codon usage for expression in E. coli was prepared for optimal expression. The gene encoding for the fusion receptor protein GST-SV2C, having a sequence denoted herein as SEQ ID NO. 11, was cloned into the expression vector pET-9a.

The plasmid pET-9a-gst-sv2c was transformed into E. coli BL21(DE3). A starter culture was prepared by inoculating a colony into 40 ml of TB media with kanamycin in an Ultra Yield 250 ml shake flask, and the flasks were incubated at 37°C, 250 rpm. After 8 hours the starter culture was used to inoculate 1 liter of TB media with kanamycin, in two Ultra Yield 2.5 liter shake flasks, and the flasks were incubated at 18°C, 250 rpm for 40 hours. The culture was harvested by centrifugation.

The cell pellet was re-suspended in 100 ml PBS and the cells were disrupted by sonication. The cell extract was clarified by centrifugation (14,000 g, 30 min) and loaded onto a GSTrap FF 5 ml column (GE Healthcare) mounted on an AKTA Explorer FPLC system (GE Healthcare). The column was washed with 15 CV of binding buffer to remove unbound proteins. The protein was eluted from the column with elution buffer (50 mM tris-HCl; 10 mM reduced glutathione, pH 8.0). The amino acid sequence of the GST- SV2C obtained is denoted herein as SEQ ID NO. 12.

Binding analysis of b-gal-Hc/A to GST-SV2C

Analysis of binding of B-gal-Hc/A to GST-SV2C was performed as follows: a 96- well plate was coated with 50 μl per well of the GST-SV2C construct diluted 1:200 in coating buffer (50 mM Na₂C0₃, pH 9.6) and incubated overnight at 4°C. The plate was then washed with wash solution (NaCl 0.9%, Tween 200.05%) and blocked for one hour at 37°C with TSTA buffer (50 mM Tris, 0.9% NaCl, 0.05% Tween, 2% BSA, 200 μl per well). After washing, the plates were incubated for one hour at 37°C with serial dilutions (from 200 to 204800) of B-gal-Hc/A in TSTA, 50 μl per well. The plate was then washed and incubated with o rlh o - n i t ro p h c n y 1 - g a 1 a c t o p y ra n o side (oNPG, sigma) solution (1 mg/ml) in Z-buffer (100 mM sodium phosphate, 10 mM KC1, 1 mM MgS0₄, 50 mM B- mercaptoethanol, pH 7.0) for one hour at 37°C. The reaction was stopped with stop solution (1 M sodium carbonate), and absorbance was measured at 420 nm. Alternatively, the fluorogenic substrate 4-methylumbellyferyl-galactopyranoside (4-MUG, sigma) (0.5 mg/ml; excitation 355 nm, emission 455 nm) was used.

Binding assay in the presence of anti BoNT antibodies A 96-well plate was coated with the GST-SV2C construct as detailed above and incubated overnight at 4°C. The plate was then washed with wash solution and blocked for one hour at 37°C with TSTA buffer. In a different 96-wells polypropylene plate, the b-gal-Hc/A construct diluted 1:6000 with TSTA was incubated for one hour at room temperature with either of the following horse antibodies (prepared by the inventors): anti-BoNT/A, anti-BoNT/B, anti-BoNT/E or naive serum, diluted 1:1650 with TSTA. The b-gal-Hc/A construct - antitoxin mixtures were then transferred into the GST-SV2C construct coated plate, 50 μl per well, and the plate was incubated for one hour at 37°C. The plate was then washed and incubated with oNPG solution in Z-buffer (prepared as detailed above) for one hour at 37°C. The reaction was stopped with stop solution, and absorbance was measured at 420 nm. The residual activity of b-gal was calculated by dividing the absorbance obtained in each one of the wells by the absorbance in a control well that did not contain antitoxin.

Screening of a compound library

The following two chimeric polypeptides, prepared as descried above, were used in the screening assay: 1. b-gal-Hc/A which is composed of a beta-galactosidase domain originating from Escherichia coli fused to the receptor binding domain of BoNT/A; and 2. GST-SV2C which is composed of glutathione- s-transferase fused to a fragment of the protein receptor of BoNT/A, namely the fourth luminal loop of the synaptic vesicle glycoprotein 2C or “SV2C”.

The screening assay was performed as follows. First, the tested compounds were mixed and incubated with the b-gal-Hc/A polypeptide construct and then the mixtures were transferred into 96-wells plates previously coated with the GST-SV2C receptor polypeptide construct. Following removal of unbound b-gal-Hc/A, the b-gal-Hc/A bound to its receptor was detected by following the enzymatic activity of beta-galactosidase, as detailed above.

The compound library FOPAC1280 (Fibrary Of Pharmacologically Active Compounds, sigma) was screened using the above screening assay. This compound library contains 1280 active compounds, each at a concentration of 10 mM in DMSO. The screening procedure performed with the FOPAC1280 library was as follows:

1. A dilution plate of the FOPAC1280 library compounds was prepared by 10-fold dilution to 1 mM with 50% DMSO 50% PBS solution. 2. A polypropylene 96-wells plate was filled (90 μl per well) with a B-gal- Hc/A solution (diluted 1:90,000 in TSTA buffer to a final concentration of 3 ng/ml).

3. The diluted compounds were mixed with the B-gal-Hc/A solution (10 μl per well) and the plate was incubated for 1 hour at 25 °C with shaking (200 rpm). Control wells were prepared as follows: 1. No compound - 100% activity; 2. No β-gal-HC/A - zero activity.

4. The mixtures of the B-gal-HC/A construct with the compounds were then transferred (50 μl per well) into 96-wells polystyrene plates that were previously coated with GST-SV2C, and the plate was incubated one hour at 37°C.

5. The plates were washed with wash solution, then a substrate solution (50 μl per well of 4-MUG 0.5 mg/ml in buffer Z) was added, and the plates were further incubated for one hour, at 37°C.

6. The reaction was stopped with a stop solution (50 μl per well) and fluorescence was measured (excitation 360 nm emission 460 nm) using a plate reader (Synergy HTX, Biotek).

For each well the residual β-gal activity was calculated using the equation: Residual activity=100x(WF-BF)/PCF

Where WF is the fluorescence of the well, BF is the fluorescence of the blank, and PCF is the fluorescence of the positive control 100% activity.

Mice

CD-1 mice, female, from Charles River, UK.

Determining the therapeutic effect of the compounds in mice

Mice (n=12) were exposed to BoNT/A by injection of four (4) LD50 doses to the left side of the peritoneum. The toxin was produced from Clostridium botulinum A strain (strains A198, obtained from the Israel Institute for Biological Research collection). Sequence analysis revealed conformity of the neurotoxin gene with serotypes 62A (GenBank accession number M30196). The toxins was prepared from concentrated supernatant of culture grown for 6 days in anaerobic culture tubes. The activity of all toxin preparations was at least 3 x 10⁵ mouse 50% lethal dose (MsLD₅₀)/ml. The mice were then injected with the examined compound at the right side of the peritoneum, namely, there were 12 mice groups, for each one of the tested inhibitory compounds. A control group was injected only with PBS. Following toxin and compounds injections animal survival was monitored.

EXAMPLE 1

High throughput screening of the compound library LOPAC1280

The inventors have developed a screening method for identifying small molecule inhibitors of BoNT/A that may be potential inhibitors of other neurotoxins that bind the SV2 receptor. Briefly, the polypeptide construct B-gal-Hc/A and the receptor polypeptide constructs GST-SV2C, prepared as described above, allow detection and quantitation of the binding between the receptor binding domain of BoNT/A and its receptor SV2C. Exemplary assays for analyzing the binding of β-gal-Hc/A to GST-SV2C and for analyzing the binding of β-gal-Hc/A to GST-SV2C in the presence of anti BoNT antibodies are detailed above.

Based on the above polypeptide constructs, a high-throughput screening (HTS) assay for binding inhibitors from a compound library was developed.

Using this assay, the compound library LOPAC1280 (Library Of Pharmacologically Active Compounds, sigma) was screened, as detailed above.

Table 1 below shows the results of residual activity for a representative screening plate of the LOPAC1280 library. Wells in column 1 (A-H) were blank wells. Wells in column 12 (E-H) were positive control (“no-compound” wells). The other wells in column 12, namely wells A12, B12, C12 , and D12 contained a neutralizing monoclonal antibody (13) and they served as a positive control for an inhibiting substance. As shown in Table 1, in most of the wells, similar residual activity values were obtained. However, a substantially lower residual activity was present in well F5 (2.3%, underlined), and therefore it contained a potential binding inhibitor. Table 1 Residual activity for a representative plate of LOPAC1280.

Based on the assays performed, the following three compounds of the LOPAC1280 library presented a high inhibition of the binding between the Hc fragment of BoNT/A and its receptor SV2C: Aurintricarboxylic acid (showing a residual activity of 3%); Benserazide (showing a residual activity of 10%); and 6-hydroxy-DL-dopa (showing a residual activity of 2%).

EXAMPLE 2

Determination of IC₅₀ for inhibiting the interaction between SV2C and BoNT/A receptor binding domain

For determining the half-maximal inhibitory concentration of the three potential inhibitors, namely Aurintricarboxylic acid, Benserazide and 6-hydroxy-DL-dopa, the assay using the polypeptide constructs β-gal-Hc/A and GST-SV2C was conducted as described above, with 3 -fold serial dilutions of the compounds (Figure 1). The three compounds exhibited typical inhibitory behavior with IC50 values of about 2, 18, and 1 mM for Aurintricarboxylic acid (Figure 1A), Benserazide (Figure IB), and 6-OH-DL- DOPA (Figure 1C), respectively. It is noteworthy that performing the in vitro assay described herein spares the use of animals for evaluating the potential of the above compounds as botulinum toxin inhibitors.

EXAMPLE 3

The therapeutic effect of the compounds following exposure to BoNT/A in-vivo

Further to the above demonstration of the inhibitory effect of the compounds Aurintricarboxylic acid, Benserazide, and 6-OH-DL-DOPA on the interaction between the receptor binding domain of BoNT/A and its receptor SV2C, evaluation of the therapeutic effect of the compounds was conducted in a mouse model.

As detailed above, mice (n=12) were exposed to BoNT/A by injection of four (4) LD50 doses to the left side of the peritoneum. The mice were then injected with the examined compound at the right side of the peritoneum. Further to preliminary safety assays, the compounds were dissolved in PBS to achieve a dose of 3.125, 12.5 and 2.5 mg per mouse for Aurintricarboxylic acid, Benserazide, and 6-OH-DL-DOPA, respectively. A control group was injected only with PBS.

Following toxin and compounds injections animal survival was monitored, as demonstrated in the survival curves shown in Figure 2A for Aurintricarboxylic acid (ATA), Figure 2B for Benserazide (benz) and in Figure 2C for 6-hydroxy-DL-DOPA (6- OH-DL-DOPA).

Interestingly, all three compounds exhibited a significant therapeutic effect. Particularly, in the case of Aurintricarboxylic acid, 11 of 12 animals survived (91.6%), and for the non-surviving animal, death was delayed from a median survival time of 20.75 hours in the control group, to 96 hours (P_value< 0.0001). For Benserazide, the median survival time was delayed from 20.75 hours in the control group to 29.8 hours in the treated group (P_Value=0.0011). Finally, for 6-OH-DL-DOPA, four animals of the treated group survived the challenge (33%) and for the non- surviving animals the median survival time was delayed from 21.2 hours in the control group to 28.7 hours in the treated group (P=0.0044).

The above results clearly suggest that the compounds Aurintricarboxylic acid, Benserazide and 6-hydroxy-DL-DOPA (6-OH-DL-DOPA) are potential inhibitors of BoNT/A, specifically the receptor binding domain thereof, and therefore may inhibit the attachment of the toxin to its cellular receptor.

EXAMPLE 4

Aurintricarboxylic acid inhibits the interaction between SV2C and the receptor binding domain of BoNT/E

To evaluate whether aurintricarboxylic acid can inhibit the interaction between SV2C and the receptor binding domain of BoNT/E, the receptor binding domain of BoNT/E (having the amino acid sequence as denoted herein by SEQ ID NO. 13) was diluted to 800 ng/ml and mixed with aurintricarboxylic acid (100 mM). Control sample contained similar concentration of the receptor binding domain of BoNT/E and buffer. Following incubation of the mixture for 1 hour at 25°C, it was transferred to a 96-well plate coated with GST-SV2C as described above. The plate was then incubated for 1 hour at 37°C and unbound Hc/E was removed by washing with wash solution. Bound Hc/E was detected using horse anti BoNT/E antibody (IIBR). Then the plate was washed and incubated 1 hour at 37°C with alkaline phosphatase-conjugated Goat anti Horse IgG antibody (Jackson ImmunoResearch). Following washing of the plate it was developed by addition of the substrate p-nitrophcnyl phosphate (1 mg/ml). Following 15 minutes, absorbance was read at 405 nm. Significant lower absorbance was obtained for the wells that contained aurintricarboxylic acid, in comparison to the control wells (Figure 3).

EXAMPLE 5

Therapeutic effect of the compounds following challenge with BoNT/E

To evaluate the therapeutic effect of the compounds against a challenge with BoNT/E, mice (n=12) were exposed to the toxin by injection of four (4) LD50 doses of BoNT/E (IIBR) to the left side of the peritoneum. The mice were then injected with the examined compound at the right side of the peritoneum. The compounds were dissolved in PBS to achieve a dose of 3.125, 12.5 and 2.5 mg per mouse for Aurintricarboxylic acid, Benserazide, and 6-OH-DL-DOPA, respectively. A control group was injected only with PBS.

Following toxin and compounds injections animal survival was monitored, as demonstrated in the survival curves shown in Figure 4A for Benserazide (benz), Figure 4B for 6-hydroxy-DL-DOPA (6-OHD) and in Figure 4C for Aurintricarboxylic acid (ATA).

All three compounds exhibited a significant therapeutic effect. In particular, full protection of treated animals was received in the case of aurintricarboxylic acid (P _value<0.0001). For benserazide, the median survival time was delayed from 7.5 hours in the control group to 10.7 hours in the treated group (P_Value=0.0006). Finally, for 6-OH- DL-DOPA, the median survival time was delayed from 7.5 hours in the control group to 9.8 hours in the treated group (P_Value=0.0002).

The above results clearly demonstrate that the compounds Aurintricarboxylic acid, Benserazide and 6-hydroxy-DL-DOPA (6-OH-DL-DOPA) are potential inhibitors of BoNT/E, specifically the receptor binding domain thereof, and therefore may inhibit the attachment of the toxin to its cellular receptor.

EXAMPLE 6

Therapeutic effect of the compounds following challenge with BoNT/F

Mice (n=12) are exposed to BoNT/F by injection of 4 LD50 of toxin to the left side of the peritoneum. The mice are also injected with the examined compound at the right side of the peritoneum. The compounds are dissolved in PBS to achieve a dose of 3.125, 12.5 and 2.5 mg per mouse for Aurintricarboxylic acid, Benserazide, and 6-OH-DL- DOPA, respectively. A control group is injected only with PBS. Following toxin and compounds injections animal survival is monitored, and survival curves are drawn for evaluating the therapeutic effect by a log-rank test.

EXAMPLE 7

Therapeutic effect of the compounds following challenge with TeNT

Mice (n=12) are exposed to TeNT by injection of 4 LD50 of toxin to the left side of the peritoneum. The mice are also injected with the examined compound at the right side of the peritoneum. The compounds are dissolved in PBS to achieve a dose of 3.125, 12.5 and 2.5 mg per mouse for Aurintricarboxylic acid, Benserazide, and 6-OH-DL-DOPA, respectively. A control group is injected only with PBS. Following toxin and compounds injections animal survival is monitored, and survival curves are drawn in order to evaluate the therapeutic effect by a log-rank test.

Claims

CLAIMS:

1. A compound having the structure (I):

wherein, each of R₁, R₂ and R₃, independently of the other, is selected from -H, -C(=0)-OH, -OH, C₁-Csalkylene and C₂-C₅alkenylene, and wherein one of said R₁, R₂ and R₃ is ortho to the -OH group; or a salt thereof for use in treating and/or preventing intoxication by at least one clostridial neuro toxin.

2. The compound for use according to claim 1, wherein each of R₁ and R₂ is -OH.

3. The compound for use according to claim 2, having the structure (II):

wherein R₃ is selected from -C(=0)-0H, -OH and C₁-Csalkylene.

4. The compound for use according to claim 1, wherein C₁-Csalkylene is substituted.

5. The compound for use according to claim 4, wherein the C₁-Csalkylene is substituted by a group selected from -OH, -NR’R ’R ” and -C(=0)-OR₄, wherein each of R’ and R” is independently selected from -H, C₁-Csalkyl and C₂-C₅alkenyl, wherein R”’ is absent or is selected from -H, C₁-Csalkyl and C₂-C₅alkenyl, and wherein R4 is -H, C₁-Csalkyl or C₂-C₅alkenyl .

6. The compound for use according to claim 5, wherein -NR’R”R” ’ is selected from -NH-, -NH2, -NHR’ and -NH2R’”, and wherein each of R’ and R”’ is as defined in claim 5.

7. The compound for use according to claim 4, wherein C₁-Csalkylene is a group having the structure (III):

OH, -NR’R”R”’ and -C(=0)-0R₄,

R4 is -H, C₁-C₅alkyl or C₂-Csalkenyl,

X is -C(=0)- and -NH-NH-C(=0)-R₆-,

R₆ is an optionally substituted C₁-Csalkylene, wherein substitution is by a group selected from -OH, -NR’R”R”’, -C(=0)-OR₄, each of R’ and R” is independently selected from -H, C₁-Csalkyl and C₂-C₅alkenyl, and wherein

R”’ is absent or is selected from -H, C₁-Csalkyl and C₂-C₅alkenyl.

8. The compound for use according to claim 7, wherein n is 1.

9. The compound for use according to claim 7, wherein n is zero.

10. The compound for use according to claim 8, wherein R₅’ is absent (namely the position substituted by R₅’ is H) and R₅ is selected as defined in claim 7.

11. The compound for use according to claim 10, wherein R₅ is -NR’R”R”’, and wherein each of R’, R” and R”’ is as defined in claim 7.

12. The compound for use according to claim 7, wherein n is 1, R₅’ is -H, R₅ is NH2 and X is -C(=0).

13. The compound for use according to claim 1, having the structure (1):

14. The compound for use according to claim 9, wherein X is -NH-NH-C(=0)-R₆- and R₆ is optionally a substituted C₂alkylene.

15. The compound for use according to claim 14, wherein the C₂alkylene is substituted by -NR’R”R”’, and wherein each of R’, R” and R”’ is as defined in claim 7.

16. The compound for use according to claim 15, having the structure (2):

17. The compound for use according to claim 1, wherein one or R₁, R₂ and R₃ is -H.

18. The compound for use according to claim 1, wherein one of R₁, R₂ and R₃ is - C(=0)-0H.

19. The compound for use according to claim 1, wherein one of R₁, R₂ and R₃ is a C₂- C₅alkenylene being optionally substituted.

20. The compound for use according to claim 17 or 18, wherein one of R₁, R₂ and R₃ is a C₂-C₅alkenylene being optionally substituted.

21. The compound for use according to claim 19 or 20, wherein the C₂-C₅alkenylene group is of structure (IV):

wherein

R₇ and R₈ together with the carbon atom to which they are bonded to form a 6- memebred ring, and wherein

R₉ is an optionally substituted aryl.

22. The compound for use according to claim 21 , wherein R7 and Rs together with the carbon atom to which they are bonded form a ring structure having the structure (V):

wherein

Rio is selected from -C(=0)-0R_a and -C(=0)-R_b, wherein each of R_a and R_b is independently selected from -H, -OH, C₁-Csalkyl, C₁-C₅hydroxyalkyl; and wherein R9 is an optionally substituted aryl.

23. The compound for use according to claim 22, wherein Rio is -C(=0)-0R_a, and R_; a is -H; wherein, R9 is an optionally substituted aryl; and wherein structure (V) is of structure (VI):

24. The compound for use according to claim 21, wherein R9 is a substituted aryl, of the structure (VII):

wherein each of R7 and Rs are as defined in claim 21, each of Rn and R12, independently of the other is selected from -H, -OH, -C(=0)- OR_f, -C(=0)-R_g and wherein each of R_f and R_g is independently selected from -H, C₁-Csalkyl and C₁- C₅hydroxyalkyl.

25. The compound for use according to claim 24, wherein R₁₁ is -OH and R₁₂ is - C(=0)-0H; the group having the structure (VII) is of the structure (VIII):

26. The compound for use according to claim 25, having the structure (3):

27. The compound for use according to any one of claims 1 to 26, wherein said clostridial neurotoxin is a Botulinum neurotoxin or a tetanus neurotoxin.

28. The compound for use according to any one of claims 1 to 26, wherein said intoxication is botulism or tetanus intoxication.

29. A compound selected from compounds herein designated compound (1), compound (2) and compound (3) for use in treating and/or preventing botulism or tetanus intoxication.

30. An inhibitor of a clostridial neurotoxin, wherein the inhibitor is any one of the compounds according to any one of claims 1 to 27.

31. The inhibitor according to claim 30, wherein said clostridial neurotoxin is a Botulinum neurotoxin, a tetanus neurotoxin or a combination thereof.

32. The inhibitor according to claim 31, wherein said Botulinum neurotoxin is Botulinum neuro toxin A (BoNT/A), Botulinum neuro toxin E (BoNT/E), Botulinum neurotoxin D (BoNT/D) or Botulinum neurotoxin F (BoNT/F) or any combination thereof.

33. A method for preventing and/or treating intoxication by a clostridial neurotoxin in a subject in need thereof, comprising administering to said subject an effective amount of at least one compound according to any one of claims 1 to 27 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

34. The method according to claim 33, wherein said intoxication by a clostridial neurotoxin is caused by at least one of BoNT/A, BoNT/E, BoNT/D, BoNT/F and TeNT.

35. The method according to claim 33, wherein said intoxication by a clostridial neurotoxin is botulism.

36. The method according to any one of claims 33 to 35, wherein said intoxication is botulism caused by at least one of BoNT/A or BoNT/E.

37. A method for inhibiting the binding of a clostridial neurotoxin to synaptic vesicle glycoprotein 2 (SV2) in a subject, said method comprising administering to said subject an effective amount of at least one compound according to any one of claims 1 to 27 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

38. The method according to claim 37, wherein the clostridial neurotoxin is at least one of BoNT/A, BoNT/E, BoNT/D, BoNT/F, and tetanus toxin.

39. The method according to any one of claims 33 to 38, wherein said subject has been exposed to Clostridium bacteria, to a Botulinum neurotoxin or to a tetanus neurotoxin.

40. The method according to any one of claims 33 to 38, wherein said subject is at risk of exposure to Clostridium bacteria , to a Botulinum neurotoxin or to a tetanus neurotoxin.

41. The method according to any one of claims 33 to 40, wherein said subject is a human.

42. The method according to any one of claims 33 to 41, wherein said intoxication by a clostridial neurotoxin is foodbome botulism, infant botulism, wound botulism or inhalation botulism.

43. The method according to any one of claims 33 to 42, wherein said at least one compound according to any one of claims 1 to 27 or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered to said subject as a single dosage unit or as multiple dosage units.

44. The method according to any one of claims 33 to 43, wherein said at least one compound according to any one of claims 1 to 27 or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered by intravenous, intramuscular, parenteral, oral, nasal, or rectal administration.

45. The method according to any one of claims 33 to 44, wherein said method further comprises administration of at least one additional therapeutic agent.

46. The method according to claim 45, wherein said of at least one additional therapeutic agent is at least one antibody directed against a clostridial neurotoxin.

47. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 27 for treating or preventing intoxication by at least one clostridial neuro toxin.