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TREATMENT OF POST-OPERATIVE SURGICAL PAIN

20230218728 · 2023-07-13

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to a clostridial neurotoxin for use in treating post-operative surgical pain in a patient, said method comprising administering to a patient a clostridial neurotoxin more than 5 days prior to surgery and wherein the clostridial neurotoxin is administered: i) intradermally; or ii) intrathecally.

    Also provided are corresponding methods of treatment and administration dosages of a clostridial neurotoxin.

    Claims

    1. A clostridial neurotoxin for use in treating post-operative surgical pain in a patient, said method comprising administering to a patient a clostridial neurotoxin more than 5 days prior to surgery and wherein the clostridial neurotoxin is administered: i) intradermally; or ii) intrathecally.

    2. A method for treating post-operative surgical pain in a patient, said method comprising administering to a patient a clostridial neurotoxin more than 5 days prior to surgery and wherein the clostridial neurotoxin is administered: i) intradermally; or ii) intrathecally.

    3. The clostridial neurotoxin for use according to claim 1 or the method according to claim 2, wherein the clostridial neurotoxin is administered 6-50 days prior to surgery; preferably 10-20 days prior to surgery.

    4. The clostridial neurotoxin for use according to claim 1 or 3 or the method according to claim 2 or 3, wherein the clostridial neurotoxin is administered 14-16 days prior to surgery, preferably about 15 days prior to surgery.

    5. The clostridial neurotoxin for use according to any one of claims 1, 3 or 4 or the method according to any one of claims 2-4, wherein the administration of the clostridial neurotoxin substantially reduces post-operative surgical pain perception by the patient and wherein the reduced post-operative surgical pain perception is maintained for 24 hours immediately following surgery.

    6. The clostridial neurotoxin for use according to any one of claims 1 or 3-5 or the method according to any one of claims 2-5, wherein substantially all reduced post-operative surgical pain perception is maintained for 3 days immediately following surgery, 5 days immediately following surgery, 7 days immediately following surgery, preferably for 8 days immediately following surgery.

    7. The clostridial neurotoxin for use according to claim 5 or 6 or the method according to claim 5 or 6, wherein said reduced level of pain perception observed at a defined time point immediately following surgery is at least 50% of the maximum level of reduced pain perception observed at any time following administration of the clostridial neurotoxin, preferably at least 75% of the maximum level of reduced pain perception observed at any time following administration of the clostridial neurotoxin.

    8. A clostridial neurotoxin for use in reducing or suppressing post-operative anxiety, said method comprising administering to a patient a clostridial neurotoxin prior to surgery, wherein the clostridial neurotoxin is administered: i) intradermally; or ii) intrathecally.

    9. A method for reducing or suppressing post-operative anxiety, said method comprising administering to a patient a clostridial neurotoxin prior to surgery, wherein the clostridial neurotoxin is administered: i) intradermally; or ii) intrathecally.

    10. The clostridial neurotoxin for use according to claim 8 or the method according to claim 9, wherein the clostridial neurotoxin is administered 5 or more days prior to surgery; preferably wherein the clostridial neurotoxin is administered more than 5 days prior to surgery.

    11. The clostridial neurotoxin for use according to claim 8 or 10 or the method according to claim 9 or 10, wherein the administration of the clostridial neurotoxin substantially reduces post-operative anxiety perception by the patient and wherein said reduced post-operative anxiety perception is maintained for 24 hours immediately following surgery.

    12. The clostridial neurotoxin for use according to claim 11 or the method according to claim 11, wherein substantially all reduced post-operative anxiety perception is maintained for 2 days immediately following surgery, for 5 days immediately following surgery, for 7 days immediately following surgery, preferably for 9 days immediately following surgery.

    13. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-12 or the method according to any one of claims 2-7 or 9-12, wherein the clostridial neurotoxin treats post-operative surgical pain and reduces or suppresses post-operative anxiety.

    14. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 13 or the method according to any one of claims 2-7 or 13, wherein said post-operative surgical pain is caused by surgical intervention and wherein the clostridial neurotoxin is administered at a site distal to the site of surgical intervention.

    15. The clostridial neurotoxin for use according to claim 14 or the method according to claim 14, wherein the distal site to the surgical incision is at least 15 cm, 50 cm or 100 cm from the site of surgical intervention.

    16. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-15 or the method according to any one of claims 2-7 or 9-15, wherein, following administration, the clostridial neurotoxin travels by retrograde transport to the spinal cord and effects SNARE protein cleavage (SNAP-25 protein cleavage) in said spinal cord.

    17. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-16 or the method according to any one of claims 2-7 or 9-16, wherein the clostridial neurotoxin is administered at an intradermal site and wherein minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) by said clostridial neurotoxin is observed at or proximal to said intradermal site following administration of the clostridial neurotoxin.

    18. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-16 or the method according to any one of claims 2-7 or 9-16, wherein the clostridial neurotoxin is administered at a site in the intrathecal space of the spinal cord and wherein minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) by said clostridial neurotoxin is observed at or proximal to said site following administration of the clostridial neurotoxin.

    19. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-18 or the method according to any one of the claims 2-7 or 9-18, wherein the surgical intervention comprises an incision to the skin and/or fascia and/or muscle, preferably wherein the surgical intervention comprises of an incision to the skin.

    20. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-19 or the method according to any one of claims 2-7 or 9-19, wherein the clostridial neurotoxin is a botulinum neurotoxin (BoNT).

    21. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-20 or the method according to any one of claims 2-7 or 9-20, wherein the clostridial neurotoxin is botulinum neurotoxin serotype A (BoNT/A).

    22. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-21 or the method according to any one of claims 2-7 or 9-21, wherein the post-operative surgical pain is acute post-operative surgical pain.

    23. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-22 or the method according to any one of claims 2-7 or 9-22, wherein the post-operative surgical pain is chronic post-operative surgical pain.

    24. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-23 or the method according to any one of claims 2-7 or 9-23, wherein said use or said method does not include intramuscular administration of the clostridial neurotoxin.

    25. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-24 or the method according to any one of claims 2-7 or 9-24, wherein the patient is administered 100-500 U of the clostridial neurotoxin; preferably wherein the patient is administered 200 U of the clostridial neurotoxin.

    26. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-25 or the method according to any one of claims 2-7 or 9-25, wherein the patient is administered a total dose of 1-3 ng of the clostridial neurotoxin.

    27. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-26 or the method according to any one of claims 2-7 or 9-26, wherein the patient is administered 80-250 pg of the clostridial neurotoxin per kg (bodyweight).

    28. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-27 or the method according to any one of claims 2-7 or 9-27, wherein the clostridial neurotoxin is administered at more than one administration site; preferably wherein the patient is administered 2.5-30 U of the clostridial neurotoxin per administration site; more preferably wherein the patient is administered 20 U of the clostridial neurotoxin per administration site.

    29. The clostridial neurotoxin for use according to any one of claims 1, 3-8 or 10-28 or the method according to any one of claims 2-7 or 9-28, wherein the clostridial neurotoxin is administered at more than one administration site; preferably wherein the patient is administered 10-170 pg of the clostridial neurotoxin per administration site; more preferably 1-14 pg/kg bodyweight per administration site.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0248] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

    [0249] FIG. 1 shows a schematic of injection sites along the surgical incision, in which Dysport, saline or the reference compound Exparel were injected.

    [0250] FIG. 2 shows a schematic of the arena used for the open field test for locomotion activity.

    [0251] FIG. 3 shows the effects from peri-operative administration of intradermal injections of either saline, Exparel (control agent) or different concentrations of Dysport (100, 200 and 400U) on mitigating post-operative surgical pain and anxiety. A Von Frey assay as a measure of surgical pain perception was conducted and results are shown in (A). The horizontal line cutting across the graph, set at 26 g, indicates the baseline for not sensing surgical pain (e.g. a threshold, above which the subject's post-operative surgical pain may be considered treated). Pain can be defined as moderate/severe when the mechanical sensitivity is 0-15 g, mild/moderate when between 15-26 g and little/no pain is sensed when above 26 g. The time taken (in seconds) for pigs to approach their handlers following peri-operative, intradermal administration of Exparel or Dysport is shown in (B). The distress behaviour was scored following peri-operative administration of Exparel or Dysport and is shown in (C).

    [0252] FIG. 4 shows the mean group total walking distance (in metres) that the animals walked in a period of 5 minutes (A) and the mean percentage of time spent in the central zone of the open field apparatus (B) following the peri-operative intradermal injection of either saline, Exparel (control agent) or different concentrations of Dysport (100, 200 and 400U).

    [0253] FIG. 5 shows a Von Frey assay of saline (control) versus BoNT/A (Dysport) intradermal administration at 15 days (A), 5 days (B) or 1 day (C) prior to surgery.

    [0254] FIG. 6 shows the latency (in seconds) of pigs to approach their handler following the intradermal administration of either saline or Dysport (200U/pig) at 15 days (A), 5 days (B) or 1 day (C) prior to surgery. For each timepoint (day) in the bar charts (A-C), the bar on the left (lighter) shows results for Dysport treatment, and the bar on the right (darker) shows results for saline treatment.

    [0255] FIG. 7 shows the distress behaviour score of pigs following the intradermal administration of either saline or Dysport at 15 days (A), 5 days (B) or 1 day (C) prior to surgery. For each timepoint (day) in the bar charts (A-C), the bar on the left (lighter) shows results for Dysport treatment, and the bar on the right (darker) shows results for saline treatment.

    [0256] FIG. 8 shows the total walking distance (in meters) of pigs following the intradermal administration of either saline or Dysport at 15 days (A), 5 days (B) or 1 day (C) prior to surgery.

    [0257] FIG. 9 shows the time course of time spent in the central zone of an open-field apparatus in a period of 5 minutes (Individual values and Median) following the intradermal administration of either saline or Dysport at 15 days (A), 5 days (B) or 1 day (C) prior to surgery.

    [0258] FIG. 10 shows a Von Frey assay of saline versus BoNT/A (Dysport) when administered via intradermal (A), subcutaneous (B) or intramuscular (C) injections. For each injection route tested, a total of 200U of Dysport per pig was administered.

    [0259] FIG. 11 shows the latency (in seconds) of pigs to approach their handler when administered saline or BoNT/A (Dysport) via intradermal (A), subcutaneous (B) or intramuscular (C) injections. For each injection route tested, a total of 200U of Dysport per pig was administered.

    [0260] FIG. 12 shows the distress behaviour score of pigs when administered saline or BoNT/A (Dysport) via intradermal (a), subcutaneous (b) or intramuscular (c) injections. For each injection route tested, a total of 200U of Dysport per pig was administered.

    [0261] FIG. 13 shows the time course of total distance (in metres) that the animals walked in a period of 5 minutes in open-field following intradermal, subcutaneous or intramuscular administration of saline or Dysport (Individual values and Mean±SEM).

    [0262] FIG. 14 shows the time course of time (percentage) spent in the central zone of an open-field apparatus following the intradermal, subcutaneous or intramuscular administration of saline or Dysport (Individual values and Mean±SEM).

    [0263] FIG. 15 shows immunohistochemistry staining of SNAP-25 in skin samples with small nerves around arterioles (A), nerve endings in the hair erector muscles (B) and small-middle sized nerves in the dermis (C).

    [0264] FIG. 16 shows immunohistochemistry staining of cleaved SNAP-25 in the spinal cord of a pig when untreated (A) and cleaved SNAP-25 staining in the ipsilateral horn (B) or contralateral horn (C) of a pig treated with Dysport.

    [0265] FIG. 17 shows the expression levels of calcitonin gene related peptide (CGRP) and Substance P in the spinal cord of a pig when either untreated (A, C) or administered an intradermal injection of Dysport (B, D).

    [0266] FIG. 18 shows the expression levels of Iba1 (A, B) and glial fibrillary acidic protein (GFAP) (C, D) in the spinal cord of a pig when untreated or when administered with intradermal injections of Dysport.

    [0267] FIG. 19 shows a Von Frey assay of either saline (control) or BoNT/A (Dysport) intradermal administration at 15 days prior to surgery or Exparel intradermal administration on the day of surgery (D1), and when pigs are subjected to a surgical incision in the left leg (A) * p<0.05; ** p<0.01; *** p<0.001; ****p<0.0001 vs. saline group using one way ANOVA followed by Tukey test. #p<0.05; ##p<0.01 ####p<0.0001 Dysport vs. Exparel group using one way ANOVA followed by Tukey test. $$ p<0.01: post-surgery timepoint vs. Day −4 using paired T-test). (B) shows the latency (in seconds) of pigs to approach their handler following the intradermal administration of either saline (control) or BoNT/A (Dysport) intradermal administration at 15 days prior to surgery or Exparel intradermal administration on the day of surgery (D1), and when pigs are subjected to a surgical incision in the left leg ($$ p<0.01: post-surgery timepoint vs. Day −4 using paired T-test. £££p<0.001: Day −16 vs. Day −4 using paired T-test. *p<0.05; ** p<0.01; ***p<0.001 treatment vs. saline group using one-way ANOVA followed by Tukey test). (C) shows the distress behaviour score of pigs following the intradermal administration of either saline (control) or BoNT/A (Dysport) intradermal administration at 15 days prior to surgery or Exparel intradermal administration on the day of surgery (D1), and when pigs are subjected to a surgical incision in the left leg (*p<0.05, ***p<0.001 and ****p<0.0001 vs. saline group using one way ANOVA followed by Tukey test. $$ p<0.01, $p<0.05: post-surgery timepoint vs. Day −4 using paired T-test).

    [0268] FIG. 20 shows a summary of the tissue samples (formalin-fixed-paraffin-embedded tissues) that were collected for immunohistochemistry staining and the specific regions where tissue samples were collected from the spinal cord.

    [0269] FIG. 21 shows the immunohistochemistry staining for cleaved SNAP-25 in the skin (A), muscle (B), and dorsal root ganglia (C) in pigs with a surgical incision in the left leg.

    [0270] FIG. 22 shows cleaved SNAP-25 staining in the ipsilateral dorsal horn of lumbar L5-L6 in the spinal cord of pigs with a surgical incision to the left leg (A) and a magnified view (B).

    [0271] FIG. 23 shows a grading scale used to determine the intensity of cleaved SNAP-25 staining. Cleaved SNAP-25 staining was graded on a scale of 1-3, with grade 0=no cleaved SNAP-25 staining, grade 1=low intensity cleaved SNAP-25 staining, grade 2=average cleaved SNAP-25 intensity staining and grade 3=high intensity cleaved SNAP-25 staining (A). FIG. 23 also shows the quantification of staining intensity for different regions of the spinal cord: lumbar L5-L6, L3-L4, L1-L2 and the thoracic and cervical regions. Staining intensity was measured by way of a “H-Score” and calculated by % of positive spinal cord sections x staining intensity in the dorsal horns (B).

    [0272] FIG. 24 provides a summary of the presence, “positive” or absence, “negative” of cleaved SNAP-25 staining in collected tissue samples.

    TABLE-US-00004 SEQUENCE LISTING Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional. SEQ ID NO: 1-BoNT/A1, accession number A5HZZ9, amino acid seauence MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLST DNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADI IQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEK YLLSEDTSGKFSVDKLKFDKLYKMLTEPYTEDNFVKFFKVLNRKTYLNFDKAVFKPNPVPKVNYTPYDGFNLRNTNPAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFE HGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPA LNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM PPYGVKRLEDFDASLKDALLKYPYDNRGTLPGQVDRLKDKVNNTLSTDPPFQLSKYVDNQRLLSTFTEYPKNPPNTSPLN LRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKP PSNLGNPHASNNPMFKLDGCRDTHRYPWPKYFNLFDKELNEKEPKDLYDNQSNSGPLKDFWGDYLQYDKPYYMLNLYDPN KYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGC SWEEPPVDDGWGERPL SEQ ID NO: 2-BoNT/B1, accession number B1INP5, amino acid seauence MPVTPNNFNYNDPPDNNNPPMMEPPFARGTGRYYKAFKPTDRPWPPPERYTFGYKPEDFNKSSGPFNRDVCEYYDPDYLN TNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLII FGPGPVLNENETPDPGPQNHFASREGFGGPMQMKFCPEYVSVFNNVQENKGASPFNRRGYFSDPALPLMHELPHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIY KNKFKDKYKFVEDSEGKYSPDVESFDKLYKSLMFGFTETNPAENYKPKTRASYFSDSLPPVKPKNLLDNEPYTPEEGFNP SDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YPENDFPPNELPLDTDLPSKPELPSENTESLTDFNVDVPVYEKQPAPKKPFTDENTPFQYLYSQTFPLDPRDPSLTSSFD DALLFSNKVYSFFSMDYPKTANKVVEAGLFAGWVKQPVNDFVPEANKSNTMDKPADPSLPVPYPGLALNVGNETAKGNFE NAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY KALNYQAQALEEPPKYRYNPYSEKEKSNPNPDFNDPNSKLNEGPNQAPDNPNNFPNGCSVSYLMKKMPPLAVEKLLDFDN TLKKNLLNYPDENKLYLPGSAEYEKSKVNKYLKTPMPFDLSPYTNDTPLPEMFNKYNSEPLNNPPLNLRYKDNNLPDLSG YGAKVEVYDGVELNDKNQFKLTSSANSKPRVTQNQNPPFNSVFLDFSVSFWPRPPKYKNDGPQNYPHNEYTPPNCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEI IFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGE ILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTPQPKEYDEQPTYSCQLLFKKDEESTDEPGLPGPHRFYESGPVFEEYKDYFCPSKWYLKEVKRKPYNLKLGCNW QFPPKDEGWTE SEQ ID NO: 3-BoNT/C1, accession number P18640, amino acid seauence MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTD SDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITG PRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG PAIPNDQTPSSVTSNPFYSQYNVKLEYAEPYAFGGPTPDLPPKSARKYFEEKALDYYRSPAKRLNSPTTANPSSFNKYPG EYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFN PPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCHKAPDGRSLYNKTLDCRELLVKNTDLPFPGDPSDVKTDPFLRK DPNEETEVPYYPDNVSVDQVPLSKNTSEHGQLDLLYPSPDSESEPLPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVE DFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISN SVRRGNFTEAFAVTGVTPLLEAFPEFTPPALGAFVPYSKVQERNEPPKTPDNCLEQRPKRWKDSYEWMMGTWLSRPPTQF NNPSYQMYDSLNYQAGAPKAKPDLEYKKYSGSDKENPKSQVENLKNSLDVKPSEAMNNPNKFPRECSVTYLFKNMLPKVP DELNEFDRNTKAKLPNLPDSHNPPLVGEVDKLKAKVNNSFQNTPPFNPFSYTNNSLLKDPPNEYFNNPNDSKPLSLQNRK NTLVDTSGYNAEVSEEGDVQLNPPFPFDFKLGSSGEDRGKVPVTQNENPVYNSMYESFSPSFWPRPNKWVSNLPGYTPPD SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIYINGKLIDTIKVKELTGI NFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYL NRYMYANSRQPVFNTRRNNNDFNEGYKPPPKRPRGNTNDTRVRGGDPLYFDMTPNNKAYNLFMKNETMYADNHSTEDPYA PGLREQTKDPNDNPPFQPQPMNNTYYYASQPFKSNFNGENPSGPCSPGTYRFRLGGDWYRHNYLVPTVKQGNYASLLEST STHWGFVPVSE SEQ ID NO: 4-BoNT/D, accession number P19321, amino acid seauence MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTD EQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFG PLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNID KYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFN LTNKGFNIENSGQNIERNPALQKLSSESVVDLETKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITL TTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALR GNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHIN YQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELN KFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALV DTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS GWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYINGELKQSQKIEDLDEVKLDKT IVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVL VQYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNY GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGWVE SEQ ID NO: 5-BoNT/E1, accession number WP_003372387, amino acid sequence MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEK DRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFET NSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVN INKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPIT GRGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFI NNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPEL LIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE SKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLNYIIQH GSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYK YPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII WTLQDNAGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTR YIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANR LYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMN FKNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK SEQ ID NO: 6-BoNT/F1, accession number Q57236, amino acid sequence MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTT DAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGP DIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGAR GVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYF QWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVN NRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLN NNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGETNISLTSSI DTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENF KEAFELLGAGILLEFVPELLIPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERFITESSIFYLMKLINEAKVSK LREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNK FIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDC IRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNSRIYINGNLIDEKSISNLGD IHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNS NFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKL IRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQEN SEQ ID NO: 7-BoNT/G, accession number WP_039635782, amino acid sequence MPVNIKNFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSKDVYEYYDPTYLK TDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLII FGPGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLY GIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYK QIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIA SKNLKTEFNGQNKAVNKEAYEEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYN TQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGDSLFEYLHAQTFPSNIENLQL TNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVKGVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETA KENFKNAFEIGGAAILMEFIPELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTI KERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKL KDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDSIPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRL IDSSGYGATMNVGSDVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTII SCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLGNANIYINGSLKKSEKILNLD RINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVSSLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKY FSKASMGETAPRTNFNNAAINYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQ LFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISENINKL RLGCNWQFI PVDEGWTE SEQ ID NO: 8-BoNT/DC, accession number BAM65681, amino acid sequence MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTD EQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFG PLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGSDVEIIPQIERLQLREKALGHYKDIAKRLNNINKTIPSSWSSNID KYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSKHYLPVFANILDDNIYTIINGFN LTTKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCLRLTRNSRDDSTCIQVKNNTLPYVADKDSISQEIFESQIITDE TNVENYSDNFSLDESILDAKVPTNPEAVDPLLPNVNMEPLNVPGEEEVFYDDITKDVDYLNSYYYLEAQKLSNNVENITL TTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSAIIPYIGPALNIGNSALR GNFKQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHIS YQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELN KFDLKTKTELINLIDSHNIILVGEVDRLKAKVNESFENTIPFNIFSYTNNSLLKDMINEYFNSINDSKILSLQNKKNTLM DTSGYNAEVRVEGNVQLNPIFPFDFKLGSSGDDRGKVIVTQNENIVYNAMYESFSISFWIRINKWVSNLPGYTIIDSVKN NSGWSIGIISNFLVFTLKQNENSEQDINFSYDISKNAAGYNKWFFVTITTNMMGNMMIYINGKLIDTIKVKELTGINFSK TITFQMNKIPNTGLITSDSDNINMWIRDFYIFAKELDDKDINILFNSLQYTNVVKDYWGNDLRYDKEYYMINVNYMNRYM SKKGNGIVFNTRKNNNDFNEGYKIIIKRIIGNTNDTRVRGENVLYFNTTIDNKQYSLGMYKPSRNLGTDLVPLGALDQPM DEIRKYGSFIIQPCNTFDYYASQLFLSSNATTNRIGILSIGSYSFKLGDDYWFNHEYLIPVIKIEHYASLLESTSTHWVF VPASE SEQ ID NO: 9-BoNT/F7, amino acid sequence MPVNINNFNYNDPINNTTILYMKMPYYEDSNKYYKAFEIMDNVWIIPERNIIGKKPSDFYPPISLDSGSSAYYDPNYLTT DAEKDRFLKTVIKLFNRINSNPAGQVLLEEIKNGKPYLGNDHTAVNEFCANNRSTSVEIKESKGTTDSMLLNLVILGPGP NILECSTFPVRIFPNNIAYDPSEKGFGSIQLMSFSTEYEYAFNDNTDLFIADPAISLAHELIHVLHGLYGAKGVTNKKVI EVDQGALMAAEKDIKIEEFITFGGQDLNIITNSTNQKIYDNLLSNYTAIASRLSQVNINNSALNTTYYKNFFQWKYGLDQ DSNGNYTVNISKFNAIYKKLFSFTECDLAQKFQVKNRSNYLFHFKPFRLLDLLDDNIYSISEGFNIGSLRVNNNGQNINL NSRIVGPIPDNGLVERFVGLCKSIVSKKGTKNSLCIKVNNRDLFFVASESSYNENGINSPKEIDDTTITNNNYKKNLDEV ILDYNSDAIPNLSSRLLNTTAQNDSYVPKYDSNGTSEIKEYTVDKLNVFFYLYAQKAPEGESAISLTSSVNTALLDASKV YTFFSSDFINTVNKPVQAALFISWIQQVINDFTTEATQKSTIDKIADISLVVPYVGLALNIGNEVQKGNFKEAIELLGAG ILLEFVPELLIPTILVFTIKSFINSDDSKNKIIKAINNALRERELKWKEVYSWIVSNWLTRINTQFNKRKEQMYQALQNQ VDGIKKIIEYKYNNYTLDEKNRLKAEYNIYSIKEELNKKVSLAMQNIDRFLTESSISYLMKLINEAKINKLSEYDKRVNQ YLLNYILENSSTLGTSSVQELNNLVSNTLNNSIPFELSEYTNDKILISYFNRFYKRIIDSSILNMKYENNRFIDSSGYGS NISINGDIYIYSTNRNQFGIYSSRLSEVNITQNNTIIYNSRYQNFSVSFWVRIPKYNNLKNLNNEYTIINCMRNNNSGWK ISLNYNNIIWTLQDTTGNNQKLVFNYTQMIDISDYINKWTFVTITNNRLGHSKLYINGNLTDQKSILNLGNIHVDDNILF KIVGCNDTRYVGIRYFKIFNMELDKTEIETLYHSEPDSTILKDFWGNYLLYNKKYYLLNLLKPNMSVTKNSDILNINRQR GIYSKTNIFSNARLYTGVEVIIRKVGSTDTSNTDNFVRKNDTVYINVVDGNSEYQLYADVSTSAVEKTIKLRRISNSNYN SNQMIIMDSIGDNCTMNFKTNNGNDIGLLGFHLNNLVASSWYYKNIRNNTRNNGCFWSFISKEHGWQE SEQ ID NO: 10-BoNT/ABmy, amino acid sequence MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD   49 TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIY   99 STDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEEL  149 NLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEES  199 LEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA  249 YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNK  299 AKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY  349 TEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAA  399 NFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKA  449 LNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLI  499 QQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYT  549 MFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATE  599 AAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK  649 DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL  699 SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINY  749 QYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNS  799 MIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDI  849 PFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVE  899 VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPK  949 YKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVF  999 FEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIA 1049 NGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKD 1099 FWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINY 1149 RDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF 1199 KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIG 1249 LIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKD 1299 EGWTE 1304 SEQ ID NO: 11-BoNT/X, amino acid sequence (GenBank: BAQ12790.1) MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLN TPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIPLPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNA TYGLYSTPISNGEGTLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGISNRNFYYNFD TGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPVQGRLGNFKLDT AEFEKKLNTILFVLNESNLAQRFSILVRKHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNA LRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPET TVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVE LTDSVDEALSNPNKVYSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNIGNDI RHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWWGTIHLQINTRLA HTYKALSRQANAIKMNMEFQLANYKGNIDDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNF DLETKKTLDKFIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGT TSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISI QDSKLIWYLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTL LDQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSDSKTITFPNNIR YGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTP YNIFHKSGLMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED SEQ ID NO: 12 (Nucleotide Sequence of Unmodified BoNT/A) ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGG TCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAG AAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAA AAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCAT TGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTC AACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAG AGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTT CGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGG CCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCA TACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTT GCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTG GTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGC GTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGT GTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATG ACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAG TTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGA CAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATT TTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTG ATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGG TCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACC TGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGT GTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACA GCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATA TTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTG CTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGT TCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGA AAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATC AACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGA ATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGT ATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTG ATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGA TAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCA ATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAG ATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTT CTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGG AGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGC GTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCT GAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACA ACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTG AATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATA CGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGT ATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATT AAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGA GTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGA GCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAAC GACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCG CAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 13 (Polypeptide Sequence of Unmodified BoNT/A) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNE KDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECK SFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDL IQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVIL LEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAII NYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTL IGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQ IQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQR VVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFII KKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGN DIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO: 14 (Nucleotide Sequence of Modified BoNT/A “Cat-A”) ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGG TCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAG AAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAA AAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCAT TGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTC AACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAG AGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTT CGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGG CCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCA TACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTT GCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTG GTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGC GTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGT GTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATG ACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAG TTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGA CAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATT TTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTG ATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGG TCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACC TGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGT GTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACA GCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATA TTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTG CTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGT TCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGA AAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATC AACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGA ATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGT ATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTG ATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGA TAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCA AGCATCTGATTGATCTGAGCCGTTATGCTAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAG ATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAAGGCCATTGTCTACAACTCCATGTACGAGAATTT CTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAAGATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGG AGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCAAAGAGATCAAGCAGCGC GTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCT GAATAAGAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACA AGATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTG AATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATA CGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGT ATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATT AAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGA GTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGA GCCAAGTCGTGGTTATGAAGAGCAAGAACGACAAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAAC GACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCG CAGCAGCcGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 15 (Polypeptide Sequence of Modified BoNT/A ″Cat-A″) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNE KDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECK SFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDL IQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVIL LEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAII NYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTL IGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESKHLIDLSRYASKINIGSKVNFDPIDKNQ IQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQR VVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFII KKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGN DIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO: 16 (Nucleotide Sequence of Modified BoNT/A “Cat-B”) ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGG TCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAG AAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAA AAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCAT TGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTC AACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAG AGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTT CGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGG CCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCA TACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTT GCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTG GTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGC GTTGATAAGCTGAAGTTTGACAAACTGTACaAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGT GTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATG ACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAG TTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGA CAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATT TTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTG ATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGG TCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACC TGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGT GTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACA GCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATA TTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTG CTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGT TCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGA AAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATC AACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGA ATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGT ATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAaTCGTGGTACGCTG ATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGA TAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCA ATCATCTGATTGATCTGAGCCGTTATGCTAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAG ATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAAGGCCATTGTCTACAACTCCATGTACGAGAATTT CTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAAGAAGATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGG AGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCAAAGAGATCAAGCAGCGC GTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCT GAATAAGAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACA AGATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTG AATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATA CGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGT ATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATT AAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGA GTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGA GCCAAGTCGTGGTTATGAAGAGCAAGAACGACAAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAAC GACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCG CAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 17 (Polypeptide Sequence of Modified BoNT/A ″Cat-B″) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNE KDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECK SFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDL IQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVIL LEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAII NYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTL IGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQ IQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFKKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQR VVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFII KKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGN DIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO: 18 (Nucleotide Sequence of Modified BoNT/A “Cat-C”) ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGG TCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAG AAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAA AAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCAT TGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTC AACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAG AGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTT CGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGG CCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCA TACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTT GCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTG GTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGC GTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGT GTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATG ACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAG TTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGA CAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATT TTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTG ATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGG TCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACC TGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGT GTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACA GCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATA TTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTG CTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGT TCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGA AAGTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATC AACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGA ATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGT ATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTG ATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGA TAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCA ATCATCTGATTGATCTGAGCCGTTATGCTAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAG ATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAAGGCCATTGTCTACAACTCCATGTACGAGAATTT CTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAAGATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGG AGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCAAAGAGATCAAGCAGCGC GTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCT GAAGAAGAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACA AGATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTG AATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATA CGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGT ATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATT AAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGA GTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGA GCCAAGTCGTGGTTATGAAGAGCAAGAACGACAAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAAC GACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCG CAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 19 (Polypeptide Sequence of Modified BoNT/A ″Cat-C″) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNE KDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECK SFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDL IQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVIL LEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAII NYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTL IGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQ IQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQR VVFKYSQMINISDYINRWIFVTITNNRLKKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFII KKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGN DIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL SEQ ID NO: 20 (Nucleotide Sequence of Modified BoNT/A “Cat-D”) ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGCCGG TCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACCCGGAAG AAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACGGATAACGAA AAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCAT TGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACTGCATTAACGTTATTC AACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAG AGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCGGATTTTACCTT CGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGG CCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACGAATGCA TACTACGAGATGAGCGGCCTgGAAGTCAGCTTCGAAGAACTGCGCACCTTCGGTGGCCATGACGCTAAATTCATTGACAGCTT GCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTTG GTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGC GTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAaGT GTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCaAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATG ACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAG TTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGA CAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATT TTACCAACGACCTGAACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTG ATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGG TCAGCTGGAACTGATGCCGAATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACC TGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGT GTCTATACCTTCTTCAGCAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACA GCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAaTTGCTGATaTTACCATCATTATCCCGTATA TTGGTCCGGCACTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTG CTGGAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGT TCAGACCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGA AAGTCaATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTATC AACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGA ATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATGATTCCGT ATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTG ATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAGTATGTTGA TAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAACCTGCGTTACGAGAGCA ATCATCTGATtGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAACCAG ATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCTACAACTCCATGTACGAGAATTT CTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGG AGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAGGACACCCAAGAGATCAAGCAGCGC GTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCT GAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGTAATATCCACGCAAGCAACA ACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTG AATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATA CGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGT ATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATT AAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGCGTAAAGA GTACCGTCTGGCGACCAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTCGTGTCCGTCGTCTGA GCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACCGTCGTGGTAAC GACATCGGCTTTATTGGTTTCCACCAGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCG CCGTAGCCGTCGTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG SEQ ID NO: 21 (Polypeptide Sequence of Modified BoNT/A ″Cat-D″) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNE KDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECK SFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTK LKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDL IQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSR VYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVIL LEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAII NYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTL IGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQ IQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQR VVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFII KKYASGNKDNIVRNNDRVYINVVVKRKEYRLATNASQAGVEKILSALEIPRVRRLSQVVVMKSKNDQGITNKCKMNLQDRRGN DIGFIGFHQFNNIAKLVASNWYNRQIERRSRRLGCSWEFIPVDDGWGERPL

    EXAMPLES

    Materials and Methods

    [0273] Animal Model

    [0274] Male domestic pigs weighing 11-13 kg were used in the following study. The pig is a suitable model for studying the treatment of post-operative surgical pain as porcine skin shares similarities with human skin in terms of structure, thickness, innervation, pigmentation, collagen and lipid composition, wound-healing and immune responses.

    [0275] Reconstitution of Dysport

    [0276] Dysport was provided in vials containing 500U. For dosing, vials of 500U were reconstituted with saline (0.9% NaCl). Subsequent dilutions were done with saline according to the testing doses as follows:

    [0277] 2.5 ml of saline was drawn with 3 ml syringe+21 G needle and transferred to the Dysport 500U vial; the concentration was 200U/ml=400U/2 ml; the vial was gently swirled until material was dissolved. Each vial was tilted side-to-side 2-3 times (to ensure solution homogeneity); pigs were dosed with 2 ml using 2 syringes of 1 ml connected to a 30 G needle. This solution was used for dosing of 400U.

    [0278] Preparation for 200U/2 ml Dose:

    [0279] Dysport was reconstituted as explained above; 3 ml syringe and 21 G needle were used to draw 2 ml of reconstituted Dysport 500U; 3 ml syringe and 21 G needle were used to draw 2 ml of saline; a vacutainer vial was used to mix the 2 solutions mentioned above; the mixed solution was tilted side-to-side 5-6 times (to ensure solution homogeneity); the pigs were dosed with 2 ml using 2 syringes of 1 ml connected to a 30 G needle.

    [0280] Preparation for 100U/2 ml Dose:

    [0281] Dysport was reconstituted as explained above; 3 ml syringe and 21 G needle were used to draw 2 ml of reconstituted Dysport 500U; 3 ml syringe and 21 G needle were used to draw 2 ml of saline; a vacutainer vial was used to mix the 2 solutions mentioned above; the mixed solution was tilted side-to-side 5-6 times (to ensure solution homogeneity); 3 ml syringe and 21 G needle were used to draw 2 ml of prepared solution from the vacutainer vial; 3 ml syringe and 21 G needle were used to draw 2 ml of saline; a new vacutainer was used to mix the 2 solutions mentioned above; the mixed solution was tilted side-to-side 5-6 times (to ensure solution homogeneity); pigs were dosed with 2 ml using 2 syringes of 1 ml connected to 30 G needle.

    [0282] Induction of Post-Operative Surgical Pain

    [0283] Pigs were anesthetized by an isoflurane/oxygen mixture, which was delivered through a facemask. The area of the incision was cleaned using Septol and Polydine (Iodo-Vit) solution. A 6-7 cm long skin incision was made in the left flank, towards the caudal end of the pig and 3 cm lateral to the spine line (Day 1) or a 7 cm long skin incision was made in the left leg. Then the fascia was cut and the muscle was retracted (Castel et al., Characterization of a porcine model of post-operative pain, Eur. J. Pain. 2014, 18(4), 496-505; incorporated herein by reference). The sub-cutis was then sutured with 3-0 Vicryl thread. The skin was sutured with 3-0 silk thread using continuous suturing methods. Following the incision closure and material injection, the pigs received antibiotic (Marbocyl 10%). The area of the incision was covered with the thin layer of Syntomicine 3%. The animals were kept under anesthesia for the duration of the surgery and dosing (about 20 minutes). Post-surgery the animals were returned to their pens for recovery and observation.

    [0284] Treatment

    [0285] Intradermal peri-operative administration of Dysport

    [0286] For peri-operative administration, animals were injected just after suturing the incision made in the left flank. Dysport (test item), saline (negative control) or the reference compound Exparel (positive control) were injected intradermally (or subcutaneously for Exparel) using 30G needles attached to 1 ml syringes and into 10 sites around the incision. Each site was injected with a fixed dosing volume and fixed dosing level. In more detail, 4 sites along each side (e.g. 8 sites) of a 7 cm horizontal incision/suture in the left flank were injected (at 2 cm intervals), as were sites at each end of the incision/suture in the left flank (see FIG. 1). The following experimental groups were assessed as follows:

    TABLE-US-00005 Group Number of Dosing level Number Treatment Animals Dosing volume per animal 1 Saline 6 200 μL/site of Saline injection 2 Exparel 6 20 ml 266 mg 3 Dysport 6 200 μL/site of 100 U injection 4 Dysport 6 200 μL/site of 200 U injection 5 Dysport 6 200 μL/site of 400 U injection

    [0287] Intradermal Pre-Operative Administration of Dysort

    [0288] For pre-operative injections, as animals were injected before the incision (either in the left flank or left leg of the pig) with a fixed total volume of 2 ml, the location of the further incision was tattooed first. Dysport (test item) or saline (negative control) were injected intradermally using 30G needles attached to 1 ml syringes and into 10 sites around the incision. Each site was injected with a fixed dosing volume and fixed dosing level. Administrations were performed either at 15 days, 5 days or 1 day prior to surgery. The following experimental groups (when an incision was made in the left flank of the pig) were assessed as follows:

    TABLE-US-00006 Dosing day vs. Group No. of Dosing level operation Number Treatment Animals Dosing volume per animal day 1 Dysport 6 200 μL/site of Saline −15 injection 2 Saline 6 200 μL/site of 200 U/pig as 10 injection sites injected with 20 U 3 Dysport 6 200 μL/site of Saline −5 injection 4 Saline 6 200 μL/site of 200 U/pig as 10 injection sites injected with 20 U 5 Dysport 6 200 μL/site of Saline −1 injection 6 Saline 6 200 μL/site of 200 U/pig as 10 injection sites injected with 20 U

    [0289] The following experimental groups (when an incision was made in the left leg of the pig) were assessed as follows:

    TABLE-US-00007 Dosing day vs. Group No. of Dosing level operation Number Treatment Animals Dosing volume per animal day 1 Saline 6 200 μL/site of Saline −15 injection 2 Dysport 5 200 μL/site of 200 U/pig as 10 injection sites injected with 20 U 3 Exparel 6 20 ml 266 mg 1

    [0290] Administration of Dysport Via Intradermal. Intramuscular or Subcutaneous Routes

    [0291] As animals were injected 15 days before the incision, the location of the further incision was tattooed. Dysport (test item) or saline (negative control) were injected using 30G needles attached to 1 ml syringes and into 10 sites around the incision. Each site was injected with a fixed dosing volume and fixed dosing level. Administrations were performed either via the intradermal, subcutaneous or intramuscular route.

    [0292] The following experimental groups were assessed as follows:

    TABLE-US-00008 Dosing day Dosing vs. Group Route of volume and operation Number Treatment administration dosing level day 1 Dysport Intramuscular 200 U/2 ml/ −15 2 Saline pig split into 10 sites of 200 μL 3 Dysport Intradermal 200 U/2 ml/ −15 4 Saline pig split into 10 sites of 200 μL 5 Dysport Subcutaneous 200 U/2 ml/ −15 6 Saline pig split into 10 sites of 200 μL

    [0293] Von Frey Assay

    [0294] Von Frey assay was performed in healthy, unoperated animals after Dysport/saline injections at 1, 2, 4 and 6 hours post-surgery on day 1 and once-daily for 10 days. Von Frey filaments (Ugo Basile, Italy) were applied at approximately ˜0.5 cm proximal to the incision line to the surface of the flank or leg skin. As the gram number of filaments increases, the force on the flanks' or legs' skin increases. The maximum force used was 60 g. Filaments were applied until the animal withdrew from the stimuli. Each filament was applied 3-5 times. If withdrawal was not achieved, a thicker filament was applied. If a withdrawal was achieved, a thinner filament was applied (thicker or thinner refers to higher/thicker or lower/thinner gram force). By alternating the filament thickness, the force required to achieve withdrawal reaction was determined and recorded. The size and force of the Von Frey filaments are outlined in the table below:

    TABLE-US-00009 Size 1.65 2.36 2.44 2.83 3.22 3.61 3.84 4.08 4.17 4.31 Force 0.008 0.02 0.04 0.07 0.16 0.40 060 1.00 1.40 2.00 (g) Filament in use Size 4.56 4.74 4.93 5.07 5.18 5.46 5.88 6.10 6.45 6.65 Force 4.00 6.00 8.00 10.0 15.0 26.0 60.0 100 180 300 (g) Filament in use

    [0295] Inclusion criteria: Animals were included in the study if the flank withdrawal force at baseline was ≥26 g (preferably 60 g). After surgery, pain (allodynia) was considered present if flank withdrawal force was ≤10 g. If the animal did not meet this criteria it was excluded from the study. One animal was excluded from the study due to relatively low threshold before operation (≤10 g).

    [0296] Animals were included in the study if the leg withdrawal force at baseline was ≥13 g. After surgery, pain (allodynia) was considered present if leg withdrawal force was ≤2 g. If the animal did not meet this criteria it was excluded from the study.

    [0297] Approaching Time test

    [0298] Prior to the dosing of pigs, the researcher who was conducting the approaching time (AT) test entered the pen for the first time. The normal behaviour of the pigs when someone entering their housing pen is moving away from the intruder and then approaching the person. The more familiar the pigs are with the person and the more comfortable they feel, the less time it takes them to approach. The latency to approach the researcher entering their home-pen was measured in seconds (cut-off time at 120 sec). This test was done in the morning, at least 1 hour post morning feeding (at 6:30 am) before the distress behaviour score and during the habituation period.

    [0299] Distress Behaviour Score

    [0300] Following incision, the behaviour of the animals changed. When approached, the animals moved away from the researcher entering their pen, tended to guard the incision side and sometimes used vocalization. This is the main phenomenon observed following this type of surgery, in rare cases the animals became restless or showed an isolation behaviour. The distress behaviour is scored from 0 (normal) to 7 (very distressed). The distress behaviour score test is performed immediately after the approaching time test. The animal general behaviour was monitored in their home pen during the morning period. The distress behaviour score also allows the overall health status of animals to be assessed. The behaviour of the animals was scored by an observer blind to the treatment, where the total score is the sum of all sections shown in the table below.

    TABLE-US-00010 Scoring Section Parameter Score Section 1 Avoiding standing (lying down) 1 Standing 0 Section 2 Avoiding walking 1 Walking 0 Section 3 Protecting the incision side while walking 1 Acting normal 0 Section 4 Moving away when approached by researcher 1 Not moving away when approached by researcher 0 Section 5 Restlessness 1 Normal 0 Section 6 Staying in isolation from other animals 1 Staying together with other animals 0 Section 7 Screaming (high vocalisation) 1 Normal vocalisation 0

    [0301] The assessment of the behaviour score was not done in a particular order but according to the animal's total spontaneous behaviour.

    [0302] Open Field Test for Locomotion Activity

    [0303] The open field is a rectangle arena 2.5 m wide and 4.7 m long. The walls of the arena are smooth and 1.6 m high. In the morning of the test, the animals from all groups were introduced to the open field individually, one at a time, for a period of 5 minutes (5). The locomotor activity of the animals was recorded using a CCTV camera and analysed with the AnyMaze software (Stoelting Co.). The open field test was performed at the end of behavioural testing performed in the pen (i.e., approaching time, distress behaviour and von Frey). After each open field experiment, the following parameters were analysed: total walking distance (m) and percentages of time spent in the center of the area (Zone E; See FIG. 2).

    [0304] Distressed animals and animals under pain normally walk closer to the walls of the pen or the open field apparatus. Animals with no distress will not hesitate to enter the center of the open field apparatus.

    Example 1

    [0305] Peri-Operative Administration of Dysport Provides a Delayed Analgesic and Anxiolytic Effect Post-Surgery (Incision on Left Flank of the Pig)

    [0306] Pigs were administered intradermal injections of either saline, Exparel (266 mg fixed doses) or different concentrations of Dysport immediately after suturing the incision made on the left flank of the pig (that is, peri-operatively). The mechanical sensitivity of pigs was measured by a von Frey assay as an assessment of treatment of post-operative surgical pain. When compared to the saline-treated group, Exparel showed an analgesic effect for a duration of 1 day but showed no effective analgesic activity afterwards. Administration of 400U of Dysport induced a moderate analgesic effect 2 days post-surgery. A greater analgesic effect was induced by 4 days post-surgery when pigs were administered either 200U or 400U of Dysport. All the concentrations of Dysport tested completely suppressed post-operative surgical pain 6 days post-operation. This suggests that Dysport provides an effective and prolonged analgesic effect for treating post-operative surgical pain. This data is illustrated in a bar chart in FIG. 3A.

    [0307] The latency of pigs to approach their handler was measured. Pigs were administered intradermal injections of either saline, Exparel or different concentrations of Dysport at the time of incision. By 2 hours post-surgery, all treatment groups showed a delay in approaching their handler. By 6 hours, intradermal administration with either 200U or 400U of Dysport reduced the time taken for pigs to approach their handler, with these effects continuing for up to 5 days post-surgery, suggesting a potential reduction in post-operative distress and anxiety-like reactivity. Pigs treated with either saline or Exparel failed to show any improvements in approaching their handler, suggesting that these treatments do not reduce post-operative distress and anxiety-like reactivity. This data is illustrated in FIG. 3B.

    [0308] The distress behaviour score of pigs was measured. Pigs administered either 100U, 200U or 400U of Dysport showed a reduction in their distress behaviour score by 2 days post-surgery, unlike saline and Exparel treated groups. This data is illustrated in FIG. 3C.

    [0309] The open field test showed there was no difference between the total distance that the animals walked prior to surgery and post-surgery following saline treatment. Treatment with Exparel or Dysport did not affect the total walking distance at 3 days post dosing, suggesting that there was no change in the animals' motor function following the surgery. This data is illustrated in FIG. 4A. Animals treated with 400U Dysport spent more time in the center of the open field apparatus, although this difference was not statistically significant (see FIG. 4B).

    Example 2

    [0310] Pre-Operative Administration of Dysport Induces a Faster Analgesic Effect and Suppresses the Emergence of Post-Operative Distress and Anxiety-Like Reactivity when a Surgical Incision is Made in the Left Flank of the Pig

    [0311] As the peri-operative administration of Dysport showed a delay in inducing an analgesic effect, the analgesic and anxiolytic effects of pre-operative administration of Dysport were measured. Pigs were administered intradermal injections of either saline or 200U of Dysport, at 15 days (see FIG. 5A), 5 days (see FIG. 5B) or 1 day (see FIG. 5C) prior to surgery (incision in the left flank of the pig). By using a Von Frey assay, the fastest analgesic effect was observed when Dysport was administered 15 days prior to surgery, where post-operative surgical pain was reduced by 1 day post-surgery. In comparison, when Dysport was administered 5 days prior to surgery, post-operative surgical pain was reduced by 5 days post-surgery.

    [0312] Pigs showed a reduced time to approach their handlers when administered intradermal injections of Dysport 15 or 5 days prior to surgery (see FIG. 6). Similarly, pigs showed a reduced distress behaviour score when administered intradermal injections of Dysport 15 or 5 days prior to surgery (see FIG. 7). The administration of Dysport 1 day prior to surgery did not induce as effective anxiolytic effects. This suggests that the pre-operative administration of Dysport 15 or 5 days prior to surgery fully prevents the emergence of post-operative distress and anxiety-like reactivity.

    [0313] None of the treatment groups (intradermal injections of Dysport 15 days, 5 days or 1 day prior to surgery) showed a difference in their post-operative total walking distance (see FIG. 8), suggesting that muscle activity was unaffected and there was no systemic spread of the toxin.

    [0314] The percentage of time spent in the center of the open-field apparatus by saline-injected animals was similar before and following the surgery. Animals treated with Dysport 15 days prior to surgery spent more time in the center of the open filed apparatus (see FIG. 9A). There was no difference in the percentage of time spent in the central zone between saline and Dysport treated animals, when administered either at 5 days or 1 day prior to surgery (see FIG. 9B and FIG. 9C).

    Example 3

    [0315] Intradermal Administration of Dysport Provides an Advantageous Route for Mitigating Post-Operative Surgical Pain and Suppressing the Emergence of Post-Operative Anxiety

    [0316] Different routes of 200U of Dysport administration (intradermal, subcutaneous and intramuscular injections) 15 days prior to surgery were assessed for their ability to induce analgesic and anxiolytic effects post-operatively. Surprisingly, intradermal administration provided for better results than the alternative routes (indeed, it was generally observed that only the intradermal route of Dysport administration showed a rapid, analgesic effect (see FIG. 10). Both the subcutaneous and intramuscular routes of Dysport administration showed little to no effects on analgesic activity. Pigs showed a reduced time to approach their handler and a reduced distress behaviour score when administered intradermal injections of Dysport 15 days prior to surgery (see FIG. 11 and FIG. 12). This suggests that the intradermal route of administration is effective at relieving post-operative surgical pain and preventing the full emergence of post-operative distress and anxiety-like reactivity.

    [0317] The walking distance recorded post-surgery was the same as that recorded prior to the surgery in all saline groups. Furthermore, treatment with Dysport and its route of administration (intradermal, subcutaneous or intramuscular routes) did not affect the total walking distance post-surgery (see FIG. 13).

    [0318] The percentage of time spent in the center of the open field apparatus by saline-injected animals was similar before and after surgery. There was no difference in the percentage of time animals spent in the center of the open field apparatus between the different administration routes (see FIG. 14).

    Example 4

    [0319] SNAP-25 Cleavage Occurs at a Site Distal to Dysport Injection, in the Ipsilateral Dorsal Horn of the Spinal Cord

    [0320] To assess the mechanism of action of Dysport (intradermal injection), immunohistochemistry was performed on both tissue samples at the site of surgical incision (left flank of the pig) and at the spinal cord. Cleaved SNAP-25 was not detected in the nerves of skin samples (see FIG. 15). Unexpectedly, cleaved SNAP-25 was visualised in the ipsilateral dorsal horn of the spinal cord (see FIG. 16), indicative of BoNT/A activity in the spinal cord and that post-operative surgical pain/anxiety control may be provided via a central effect in the spinal cord. This also highlights that Dysport may be administered directly into the spinal cord via intrathecal administration.

    [0321] The expression levels of two neuropeptides involved in pain modulation, calcitonin gene related peptide (CGRP) and Substance P, were assessed in the spinal cord by immunohistochemistry staining. Neither neuropeptide showed a difference in their expression level in the spinal cord of pigs treated with Dysport compared to untreated (see FIG. 17).

    [0322] The expression levels of a marker of microglial cell activation, Iba1, were decreased in the spinal cord of pigs treated with Dysport when compared to untreated (see FIG. 18A, B). Similarly, expression levels of a marker of astrocyte activation, GFAP, were decreased in the spinal cord of pigs treated with Dysport when compared to untreated (FIG. 18C, D).

    Example 5

    [0323] Pre-Operative Administration of Dysport Induces a Fast Analgesic Effect and Suppresses the Emergence of Post-Operative Distress and Anxiety-Like Reactivity when a Surgical Incision is Made in the Left Leg of the Pig

    [0324] Analgesic and anxiolytic effects of pre-operative administration of Dysport were measured when a different site of surgical incision was made to the pig (surgical incision to the left leg instead of the left flank). Pigs were administered intradermal injections of either saline or 200U of Dysport at 15 days prior to surgery or Exparel on the day of the surgery (day 1) (see FIG. 19A), (surgical incision to the left leg). By using a Von Frey assay, a fast analgesic effect was observed, where post-operative surgical pain was reduced by 1 day post-surgery and a long-lasting reversal of mechanical allodynia was observed by day 4.

    [0325] Pigs (with a sutured incision in the left leg) showed a reduced time to approach their handlers when administered intradermal injections of Dysport 15 days prior to surgery when compared to administration of saline and Exparel (see FIG. 19B). Similarly, pigs (with a sutured incision in the left leg) showed a reduced distress behaviour score when administered intradermal injections of Dysport 15 days prior to surgery when compared to administration of saline and Exparel (see FIG. 19C). This suggests that the pre-operative administration of Dysport 15 days prior to surgery (incision to the left leg) fully prevents the emergence of post-operative distress and anxiety-like reactivity.

    [0326] Overall, this experiment provides further support for the fast analgesic and anxiolytic effects when Dysport is administered 15 days prior to surgery.

    Example 6

    [0327] SNAP-25 Cleavage Occurs in the Ipsilateral Dorsal Horn of the Spinal Cord in Pigs with a Surgical Incision to the Left Leg

    [0328] To assess whether a same mechanism of action of Dysport occurred when Dysport was intradermally administered at a different site in the pig, immunohistochemistry was performed on tissue samples at the site of surgical incision (left leg of the pig) and at different regions of the spinal cord (see FIG. 20). Cleaved SNAP-25 was not detected in the nerves of skin samples on samples collected 5-7 days after the incision and injection of Dysport (see FIG. 21). Cleaved SNAP-25 was visualised in the ipsilateral dorsal horn of the spinal cord, specifically in lumbar regions L5-L6 (see FIG. 22), similar to findings in pigs with a surgical incision made to their left flank. These findings are indicative of BoNT/A activity in the spinal cord and that post-operative surgical pain/anxiety control may be provided via a central effect in the spinal cord. The localisation of cleaved SNAP-25 staining in the ipsilateral dorsal horn was different when compared to cleaved SNAP-25 staining in pigs with a surgical incision made to their left flank.

    [0329] The intensity of cleaved SNAP-25 staining was graded on a scale of 1-3, with grade 0=no cleaved SNAP-25 staining, grade 1=low intensity cleaved SNAP-25 staining, grade 2=average cleaved SNAP-25 intensity staining and grade 3=high intensity cleaved SNAP-25 staining (see FIG. 23A). Based on said grading system, pigs with a surgical incision made to their left leg had lower intensity cleaved SNAP-25 staining in the ipsilateral dorsal horn when compared to pigs with a surgical incision made to their left flank.

    [0330] The intensity of cleaved SNAP-25 staining was quantified (see FIG. 23B). A H-Score was calculated as a measure of cleaved SNAP-25 staining intensity. The H-Score was calculated by multiplying the % of positive spinal cord sections by the staining intensity in the dorsal horns. In pigs treated with Dysport (and with a surgical incision in the left leg), the highest staining of cleaved SNAP-25 was observed in the spinal cord in lumbar regions L5-L6 (assigned a cleaved SNAP-25 intensity staining of grade 2) when compared to lumbar regions L3-L4 and L1-L2, and the thoracic and cervical regions of the spinal cord (the cervical region had traces of cleaved SNAP-25 staining). There was no evidence of cleaved SNAP-25 staining in saline or Exparel injected pigs.

    [0331] The above immunohistochemistry staining is summarised in FIG. 24 and confirm that SNAP-25 cleavage is observed in localised regions of the spinal cord. Localised regions L5-L6, L3-L4 and L1-L2, and the thoracic and the cervical regions of the spinal cord tested positive for cleaved SNAP-25 staining whilst the remaining tissues (including the skin, at the injection site) tested negative for cleaved SNAP-25 staining.

    [0332] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.