Therapeutic and Cosmetic Uses of Botulinum Neurotoxin Serotype E

Abstract

The present invention is directed to a botulinum neurotoxin serotype E (BoNT/E) for use in treating a disorder, wherein the BoNT/E is administered to a human subject, and provides a maximum inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration; and reduces to >25% inhibition of neurotransmitter secretion from the target cell or tissue at day 14 after administration.

Claims

1. A botulinum neurotoxin serotype E (BoNT/E) for use in treating a disorder, wherein the BoNT/E is administered to a human subject, and provides a maximum inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration; and reduces to >25% inhibition of neurotransmitter secretion from the target cell or tissue at day 14 after administration.

2. A method for treating a disorder, the method comprising administering a BoNT/E to a human subject, and wherein the administering a BoNT/E provides a maximum inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration; and reduces to >25% inhibition of neurotransmitter secretion from the target cell or tissue at day 14 after administration.

3. A cosmetic method comprising administering a BoNT/E to a human subject, wherein the administering a BoNT/E provides a maximum inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration; and reduces to >25% inhibition of neurotransmitter secretion from the target cell or tissue at day 14 after administration.

4. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is free from complexing proteins that are present in naturally occurring BoNT/E.

5. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is recombinant BoNT/E.

6. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the total dose of BoNT/E administered is greater than 3.6 ng.

7. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the total dose of BoNT/E administered is greater than 4 ng.

8. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides: at least 40% inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration; and reduces to 15% or less inhibition of neurotransmitter secretion from the target cell or tissue in >21 to <100 days after administration.

9. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides: at least 40% inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration; and reduces to 15% or less inhibition of neurotransmitter secretion from the target cell or tissue in >40 to <80 days after administration.

10. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides a substantially linear decrease in % inhibition of neurotransmitter secretion subsequent to maximum inhibition of neurotransmitter secretion.

11. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides at least 40% inhibition of neurotransmitter secretion from the target cell or tissue in 10 days from administration.

12. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides 15% or less inhibition of neurotransmitter secretion from the target cell or tissue in <80 days after administration.

13. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides 15% or less inhibition of neurotransmitter secretion from the target cell or tissue in <60 days after administration.

14. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides 15% or less inhibition of neurotransmitter secretion from the target cell or tissue in <30 days after administration.

15. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides greater than 15% inhibition of neurotransmitter secretion for 5-100 days.

16. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides greater than 15% inhibition of neurotransmitter secretion for 5-60 days.

17. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides greater than 15% inhibition of neurotransmitter secretion for 5-40 days.

18. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides greater than 15% inhibition of neurotransmitter secretion for 5-30 days.

19. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides 40-60% inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration.

20. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides 40-60% inhibition of neurotransmitter secretion from a target cell or tissue in ≤11 days after administration.

21. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides: a. maximal inhibition of neurotransmitter secretion from a target cell or tissue in ≤13 days after administration; and b. a subsequent reduction in the % inhibition of neurotransmitter secretion of 1-10% per day.

22. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E is administered and provides: a. maximal inhibition of neurotransmitter secretion from a target cell or tissue in ≤10 days after administration; and b. a subsequent reduction in the % inhibition of neurotransmitter secretion of 1-3% per day from day 10-24, from day 6-42, from day 10-42 and/or from day 10-56 after administration.

23. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the % inhibition of neurotransmitter secretion from a target cell or tissue is measured by means of electrophysiology.

24. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E: a. is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1; or b. comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3.

25. The botulinum neurotoxin for use, method, or cosmetic method according to any one of the preceding claims, wherein the BoNT/E comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2, with the proviso that the polypeptide sequence includes one or more of the following amino acids (wherein the amino acid position numbering starts with the N-terminal methionine amino acid residue and ends with the C-terminal amino acid residue of the BoNT/E protein): glycine at position 177; serine at position 198; alanine at position 340; leucine at position 773; leucine at position 963; glutamine at position 964; alanine at position 967; asparagine at position 1195.

26. The botulinum neurotoxin for use, method or according to any one of the preceding claims, wherein the disorder is one or more of: a condition associated with strabismus, blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), beauty therapy (cosmetic) applications benefiting from cell/muscle incapacitation (via SNARE down-regulation or inactivation), neuromuscular disorder or condition of ocular motility (e.g. concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, charley horse, tension headaches, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, chronic neurogenic inflammation, and a smooth muscle disorder.

27. The botulinum neurotoxin for use, or method according to any one of claim 1-2 or 4-26, wherein the disorder is upper limb spasticity.

28. The botulinum neurotoxin for use, method or use according to any one of claim 1-2 or 4-27, wherein the disorder is upper limb spasticity in a subject over the age of 18 years.

29. The cosmetic method according to any one of claim 3-25, wherein the cosmetic method is for treating an upper facial line; optionally wherein the upper facial line is one or more selected from a glabellar line, a lateral canthal line, a frontalis line, or a combination thereof.

30. The cosmetic method according to any one of claim 3-25 or 29, wherein the cosmetic method is for treating glabellar lines.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0265] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0266] FIG. 1 shows (A) the location of the extensor digitorum brevis muscle, and (B) a representation of the recorded action potential.

[0267] FIG. 2 shows the BoNT/E dose escalation procedure and results.

[0268] FIG. 3 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 12 week period following administration of different doses of rBoNT/E compared to placebo (mean+/−s.e.m.).

[0269] FIG. 4 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 26 week period following administration of different doses of Dysport® compared to placebo (mean+/−s.e.m.).

[0270] FIG. 5 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 26 week period following administration of different doses of rBoNT/E compared to placebo (mean+/−s.e.m.).

[0271] FIG. 6 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 7 day period following administration of different doses of rBoNT/E compared to placebo (mean+/−s.e.m.).

[0272] FIG. 7 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 7 day period following administration of different doses of Dysport® compared to placebo (mean+/−s.e.m.).

[0273] FIG. 8 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 26 week period following administration of 0.04 ng of rBoNT/E or 20 U Dysport® (mean+/−s.e.m.).

[0274] FIG. 9 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 7 day period following administration of 0.04 ng of rBoNT/E or 20 U Dysport® (mean+/−s.e.m.).

[0275] FIG. 10 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 26 week period following administration of 0.2 ng of rBoNT/E or 40 U Dysport® (mean+/−s.e.m.).

[0276] FIG. 11 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over a 7 day period following administration of 0.2 ng of rBoNT/E or 40 U Dysport® (mean+/−s.e.m.).

[0277] FIG. 12 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over days 10-21 following administration of 0.04 ng of rBoNT/E (mean+/−s.e.m.). A linear curve has been fit to the CMAP values (Microsoft Excel), which has an R-squared value of 0.9642, and a slope of y=3.5531x+9.9523. The CMAP values correspond to those shown for this dose in FIG. 3.

[0278] FIG. 13 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over days 10-42 following administration of 0.9 ng of rBoNT/E (mean+/−s.e.m.). A linear curve has been fit to the CMAP values (Microsoft Excel), which has an R-squared value of 0.9926, and a slope of y=1.6167x +4.8227. The CMAP values correspond to those shown for this dose in FIG. 3.

[0279] FIG. 14 shows the % of baseline compound muscle action potential (CMAP) of the stimulated extensor digitorum brevis muscle over days 10-56 following administration of 3.6 ng of rBoNT/E (mean+/−s.e.m.). A linear curve has been fit to the CMAP values (Microsoft Excel), which has an R-squared value of 0.9736, and a slope of y=1.7325x −9.3108. The CMAP values correspond to those shown for this dose in FIG. 3.

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 may be optional. (Nucleotide Sequence of BoNT/E) SEQ ID NO: 1    1 atgccaaaaa ttaatagttt taattataat gatcctgtta atgatagaac aattttatat   61 attaaaccag gcggttgtca agaattttat aaatcattta atattatgaa aaatatttgg  121 ataattccag agagaaatgt aattggtaca accccccaag attttcatcc gcctacttca  181 ttaaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtga tgaagaaaag  241 gatagatttt taaaaatagt cacaaaaata tttaatagaa taaataataa tctttcagga  301 gggattttat tagaagaact gtcaaaagct aatccatatt tagggaatga taatactcca  361 gataatcaat tccatattgg tgatgcatca gcagttgaga ttaaattctc aaatggtagc  421 caagacatac tattacctaa tgttattata atgggagcag agcctgattt atttgaaact  481 aacagttcca atatttctct aagaaataat tatatgccaa gcaatcacgg ttttggatca  541 atagctatag taacattctc acctgaatat tcttttagat ttaatgataa tagtatgaat  601 gaatttattc aagatcctgc tcttacatta atgcatgaat taatacattc attacatgga  661 ctatatgggg ctaaagggat tactacaaag tatactataa cacaaaaaca aaatccccta  721 ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggagg tactgattta  781 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc tgattataaa  841 aaaatagcgt ctaaacttag caaagtacaa gtatctaatc cactacttaa tccttataaa  901 gatgtttttg aagcaaagta tggattagat aaagatgcta gcggaattta ttcggtaaat  961 ataaacaaat ttaatgatat ttttaaaaaa ttatacagct ttacggaatt tgatttagca 1021 actaaatttc aagttaaatg taggcaaact tatattggac agtataaata cttcaaactt 1081 tcaaacttgt taaatgattc tatttataat atatcagaag gctataatat aaataattta 1141 aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta gaattattac accaattaca 1201 ggtagaggac tagtaaaaaa aatcattaga ttttgtaaaa atattgtttc tgtaaaaggc 1261 ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgt ggcttccgag 1321 aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatac agtaacttca 1381 aataataatt atgaaaatga tttagatcag gttattttaa attttaatag tgaatcagca 1441 cctggacttt cagatgaaaa attaaattta actatccaaa atgatgctta tataccaaaa 1501 tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaact taatgtattt 1561 ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatct cacctcttca 1621 attgatacag cattattaga acaacctaaa atatatacat ttttttcatc agaatttatt 1681 aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggataca acaagtgtta 1741 gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgc agatatttct 1801 atagttgttc catatatagg tcttgcttta aatataggaa atgaagcaca aaaaggaaat 1861 tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttga acccgagctt 1921 ttaattccta caattttagt attcacgata aaatcttttt taggttcatc tgataataaa 1981 aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaa atggaaagaa 2041 gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatt taataaaaga 2101 aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg caattaaaac aataatagaa 2161 tctaagtata atagttatac tttagaggaa aaaaatgagc ttacaaataa atatgatatt 2221 aagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataa tatagacagg 2281 ttcttaactg aaagttctat atcctattta atgaaattaa taaatgaagt aaaaattaat 2341 aaattaagag aatatgatga gaatgtcaaa acgtatttat tgaattatat tatacaacat 2401 ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaactga taccctaaat 2461 aatagtattc cttttaagct ttcttcttat acagatgata aaattttaat ttcatatttt 2521 aataaattct ttaagagaat taaaagtagt tcagttttaa atatgagata taaaaatgat 2581 aaatacgtag atacttcagg atatgattca aatataaata ttaatggaga tgtatataaa 2641 tatccaacta ataaaaatca atttggaata tataatgata aacttagtga agttaatata 2701 tctcaaaatg attacattat atatgataat aaatataaaa attttagtat tagtttttgg 2761 gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaata cactataata 2821 aattgtatga gagataataa ttcaggatgg aaagtatctc ttaatcataa tgaaataatt 2881 tggacattgc aagataatgc aggaattaat caaaaattag catttaacta tggtaacgca 2941 aatggtattt ctgattatat aaataagtgg atttttgtaa ctataactaa tgatagatta 3001 ggagattcta aactttatat taatggaaat ttaatagatc aaaaatcaat tttaaattta 3061 ggtaatattc atgttagtga caatatatta tttaaaatag ttaattgtag ttatacaaga 3121 tatattggta ttagatattt taatattttt gataaagaat tagatgaaac agaaattcaa 3181 actttatata gcaatgaacc taatacaaat attttgaagg atttttgggg aaattatttg 3241 ctttatgaca aagaatacta tttattaaat gtgttaaaac caaataactt tattgatagg 3301 agaaaagatt ctactttaag cattaataat ataagaagca ctattctttt agctaataga 3361 ttatatagtg gaataaaagt taaaatacaa agagttaata atagtagtac taacgataat 3421 cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaac tcacttattt 3481 ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaat atcatcatct 3541 ggcaatagat ttaatcaagt agtagttatg aattcagtag gaaataattg tacaatgaat 3601 tttaaaaata ataatggaaa taatattggg ttgttaggtt tcaaggcaga tactgtagtt 3661 gctagtactt ggtattatac acatatgaga gatcatacaa acagcaatgg atgtttttgg 3721 aactttattt ctgaagaaca tggatggcaa gaaaaataa (Polypeptide Sequence of BoNT/E) SEQ ID NO: 2 PKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPE RNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGI LLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFET NSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSL HGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLL ADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSF TEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLN PRIITPITGRGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINT PKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSD IEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKP VQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDA LELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVY SFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDI KQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLNYII QHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMR YKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKN FSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTLQDNAGINQK LAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNIL FKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYL LNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKND QVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMNFKNN NGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK (BoNT/E-UniProt Q00496) SEQ ID NO: 3 MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGS IAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKL SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE SKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKIN KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYF NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNF KNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK (Polypeptide Sequence of BoNT/E L-Chain) SEQ ID NO: 4 PKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPE RNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGI LLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFET NSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSL HGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLL ADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSF TEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLN PRIITPITGRGLVKKIIRFCKNIVSVKGIR (Polypeptide Sequence of BoNT/E H-Chain) SEQ ID NO: 5 KSICIEINNGELFFVASENSYNDDNINT PKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSD IEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKP VQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDA LELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVY SFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDI KQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLNYII QHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMR YKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKN FSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTLQDNAGINQK LAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNIL FKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYL LNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKND QVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMNFKNN NGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

EXAMPLES

[0280] Materials & Methods

[0281] Pharmacodynamic Model

[0282] The pharmacodynamic model utilised the Extensor Digitorum Brevis (EDB) muscle (FIG. 1A). The EDB is a superficial muscle of the foot, accessible for electrophysiological studies. It is involved in the extension of the 2nd to 4th digits of the foot, and weakness thereof does not impair walking.

[0283] The EDB muscle was previously used to determine the effect of BoNT/A as published in Hamjian and Walker (1994), Muscle Nerve, 17(12): 1385-92. Since then, the model has been widely used for: [0284] Quantifying onset and degree of human muscle relaxation and duration of effects following BoNT injection (Sloop et al (1996), Neurology, 46(5), 1382-6); [0285] Comparing different subtypes of BoNT (e.g. BoNT/B vs. BoNT/A) (Sloop et al (1997), Neurology, 49(1), 189-94); [0286] Checking potency of BoNT/A formulations (Park and Ahn (2013), J Clin Neurol, 9(3), 157-164).

[0287] The method involved the stimulation of the injected EDB and recording of its action potential (FIG. 1B). BoNT/E or BoNT/A was administered by way of ultrasound-guided administration and surface electrodes were used for stimulation and recording of action potentials.

[0288] FIG. 2 summarises the dose escalation procedure, which was based on review of safety (blinded) and pharmacodynamic (anonymized) data from a previous cohort.

[0289] Study Design and Treatment

[0290] Phase 1, randomised, double-blind, placebo-controlled study (human clinical trial) was conducted.

[0291] Each subject received a single intramuscular dose of rBoNT-E or placebo (randomised 3:1) into their right EDB muscle.

[0292] Cohorts of subjects were used, with each cohort injected with a higher dose of rBoNT-E than the previous. All subjects in each cohort received the same dose of rBoNT-E, or a placebo.

[0293] There was a maximum of eight cohorts: [0294] Initial cohorts: four subjects per cohort (three rBoNT-E, one placebo). [0295] Later cohorts: Once ˜50% inhibition of CMAP from baseline was achieved at three consecutive timepoints in at least of three rBoNT-E-injected subjects, cohorts were doubled to eight subjects.

[0296] Cohorts were injected sequentially in order of ascending doses: [0297] Doses were escalated until maximal CMAP inhibition (85% reduction from baseline) was achieved at three consecutive timepoints in at least four of six rBoNT-E-injected subjects per cohort, in two consecutive cohorts. [0298] Doses used were: 0.1 cell-based assay units (CBA U; 0.04 ng), 0.5 CBA U (0.2 ng), 2.25 CBA U (0.9 ng) and 9.0 CBA U (3.6 ng).

[0299] Inclusion criteria included: [0300] Healthy male adults (18 to 55 years), with body mass index 18 to 30 kg/m.sup.2. [0301] EDB CMAP total amplitude of mV by stimulating peroneal nerve for electrophysiological examination (at screening and prior to injection). [0302] Willing to comply with the study protocol and remain at the clinic for the required duration.

[0303] Exclusion criteria included: [0304] Previous BoNT treatment months prior to enrolment. [0305] History of hypersensitivity to components of rBoNT-E formulation. [0306] Medical conditions that put the subject at risk with BoNT exposure. [0307] Use of neuromuscular transmission agents.

[0308] Cell-Based Assay

[0309] The biological activity of rBoNT/E was determined with a cell-based assay (CBA) and is expressed in % relative potency to a defined reference standard. This CBA mimics the in vivo mechanism of action of BoNT/E, i.e. binding to receptors leading to internalisation of the toxin and cleavage of the synaptosomal-associated protein 25 (SNAP25) by the light chain endopeptidase domain. The CBA uses Neuro-2A, a murine neuroblastoma cell line, engineered to express a reporter which utilises a full-length SNAP-25 flanked by fluorophores, cyan fluorescent protein and yellow fluorescent protein (YFP). When the engineered cells are incubated with BoNT/E the toxin binds, and is internalised via receptor-mediated endocytosis, leading to release of the BoNT/E light chain into the cytosol. The endopeptidase light chain cleaves SNAP-25 in the reporter resulting in the release of a C-terminal reporter fragment into the cytosol that contains residues of SNAP-25 and YFP. The fragment is rapidly degraded, resulting in a loss of YFP fluorescence. Recombinant BoNT/E bioactivity is only detected if the toxin enters the cells through the BoNT/E H-chain receptor binding, internalisation, and translocation activities; thus, the in vitro CBA mimics the natural cell biology of BoNT/E. The CBA units corresponding to the amounts of BoNT/E are provided in the following Examples.

[0310] 1 CBA unit may be defined as the amount of BoNT/E required to cleave 1 nM of substrate (SNAP-25) in 1 minute.

Example 1

[0311] Administration of BoNT/E

[0312] FIG. 3 shows that the placebo varies around the 100% CMAP baseline, and shows a clear dose-effect for BoNT/E. The two higher doses showed the same pharmacodynamic (PD) profile with maximal inhibition (more than 90%) reached, and a duration of action of approximately 50 days (15% inhibition cut-off) or 30 days (50% inhibition cut-off).

[0313] Administration of Dysport®

[0314] FIG. 4 again shows the placebo varying around 100% baseline, and a 60-70% inhibition reached for the doses of 40 and 70 U of Dysport® (BoNT/A). The inhibition plateaued for several weeks with no recovery (return to 15% inhibition) at the end of the 26-week follow-up period.

Example 2

[0315] Analysis up to 26 Weeks Post-Administration

[0316] A comparison of FIGS. 4 and 5 shows that for the dose ranges investigated rBoNT-E had a faster onset of effect, greater peak effect and shorter duration of effect.

Example 3

[0317] Onset of Action

[0318] The time to onset of action (defined as first timepoint where 15% CMAP inhibition recorded) for BoNT/E was fast and consistent in all subjects and all rBoNT-E dose levels investigated. The onset was also found to range between Day 1 (day of BoNT/E administration) and Day 2 versus Day 1 to Day 7 with Dysport®. Results are presented in FIGS. 6 and 7 and values summarised in the tables below.

[0319] Table 1 shows data for rBoNT/E.

TABLE-US-00005 0.1 CBA unit 0.5 CBA unit 2.25 CBA units 9 CBA units (0.04 ng) (0.2 ng) (0.9 ng) (3.6 ng) Dose Level n = 3 n = 6 n = 6 n = 6 Time to onset (Day)  1.7 ± 0.6 1.7 ± 0.5 1.2 ± 0.4  1.5 ± 0.5 [1-2] [1-2] [1-2] [1-2] Time to reach maximal  9.0 ± 1.7 8.8 ± 4.8 5.8 ±2 .5  7.8 ± 2.4 inhibition (Day)  [7-10] [2-14] [3-10]  [5-10] Maximal inhibition (%) 53.7 ± 9.1 72.3 ± 14.9 91.5 ± 7.6  95.8 ± 3.1 [43.7-61.6] [45.1-87.8] [77.6-100] [91.6-98.7] Time to recovery (Day) 21.0 ± 0.0 37.0 ± 9.3  57.4 ± 15.2 54.6 ± 7.7 [21-21] [24-49] [42-77] [42-63] Duration of effect (days) 19.5 ± 0.7 35.2 ± 9.6  56.2 ± 15.1 53.0 ± 8.0 [19-20] [22-48] [41-76] [40-62]

[0320] rBoNT/E was associated with a fast onset of action between Day 1 (day of administration) and Day 2. Time to maximal effect was recorded between Day 2 and Day 14 (at the latest). rBoNT/E was also associated with a short duration of action lasting around 50 days for the two highest tested doses.

[0321] Table 2 shows data for Dysport®.

TABLE-US-00006 Dysport 20 U Dysport 46 U Dysport 70 U Dose level (n = 6) (n = 6) (n = 6) Time to onset (Day) 3.5 ± 2.1 2.3 ± 1.0  2.7 ± 1.9 [1-7] [1-4] [1-6] Time to reach maximal 56.5 ± 42.9 29.3 ± 19.3 15.3 ± 6.7 inhibition (Day)  [21-133]  [5-56]  [6-24] Maximal inhibition 52.2 ± 13.3 72.5 ± 12.8 70.3 ± 7.8 (%) [36.4-70.6] [58.4-92.1] [57.1-77.9] Time to recovery >Day 182 [a] >Day 182 [a] >Day 182 [a] (Day) [a] CMAP inhibition did not recovered 15% baseline at the end of the 6-month follow-up period

[0322] Dysport® exhibited a later onset of action between Day 1 and Day 7 for the dose range investigated, and a duration of action beyond 26-week post-dose.

Example 4

[0323] A Comparison of 40 pg rBoNT/E and 20 U Dysport®

[0324] FIGS. 8 and 9 show that 40 pg rBoNT-E leads to the same amplitude of effect as 20 U Dysport®. Notably, 20 U Dysport=108 pg neurotoxin (5.38 pg neurotoxin/U).

[0325] A comparison of the PD properties are compared in the table below.

[0326] Table 3 shows comparative data for rBoNT/E and Dysport®.

TABLE-US-00007 PD parameter rBoNT-E - 40 pg Dysport 20 U Time to onset D 1-D 2 D 1-D 7 Maximal effect ~50% inhibition on ~50% inhibition on average average Time to max effect  9 days on average 57 days on average Duration of effect 20 days on average >6 months on average * * about 40 days for 2/6 subjects > 26 weeks for 4/6 subjects

Example 5

[0327] A Comparison of 200 pg rBoNT/E and 40 U Dysport®

[0328] FIGS. 10 and 11 show that 200 pg rBoNT-E leads to the same amplitude of effect as 40 U Dysport®. A comparison of the PD properties are compared in the table below.

[0329] Table 4 shows comparative data for rBoNT/E and Dysport®.

TABLE-US-00008 PD parameter rBoNT-E - 200 pg Dysport 40 U Time to onset D 1-D 2 D 1-D 4 Maximal effect ~70% inhibition on ~70% inhibition on average average Time to max effect  9 days on average 29 days on average Duration of effect 35 days on average >6 months on average

[0330] In conclusion, BoNT/E exhibited: [0331] a good overall safety profile for single intramuscular doses of rBoNT-E up to 3.6 ng in healthy volunteers (normal muscle); [0332] no local diffusion to adjacent muscles (AH and ADQ); [0333] a fast onset of action irrespective of the investigated dose; [0334] a dose dependent amplitude of effect with maximal effect reached from 0.9 ng; [0335] a maximal effect reached within approximately one week; and [0336] a similar PD profile for the two highest tested doses associated with a duration of effect of approximately 50 days.

[0337] In contrast, inhibition of the CMAP was still observed at the end of the 26-week (6 months) recording period for Dysport®.

[0338] In respect of the amplitude of maximal effect: [0339] 20 U Dysport compares to 0.04 ng rBoNT-E; and [0340] 40-70 U Dysport compares to 0.2 ng rBoNT-E

Example 6

[0341] Treatment of Upper Facial Lines

[0342] A subject with severe glabellar lines (grade 3 according to the Facial Wrinkle Scale (FWS), where 0=none and 3=severe) is presented. A dose of at least 0.1 ng (per injection site) of rBoNT/E is injected intramuscularly at five sites (sequentially, such that each muscle is injected at substantially the same time): one in the procerus muscle, and two in each corrugator supercilii muscle.

[0343] The severity of glabellar lines in the subject is assessed at 7 days (after administration) using the FWS, and a grade of 0 is determined (therefore, the rBoNT/E is demonstrated to have a fast onset of action for treating upper facial lines).

[0344] The severity of glabellar lines in the subject is assessed at 35 days (after administration) using the FWS, and a grade of 2 (moderate) is determined.

[0345] The severity of glabellar lines in the subject is assessed at 56 days (after administration) using the FWS, and a grade of 3 (severe) is determined (therefore, the rBoNT/E is demonstrated to have a short duration of effect for treating upper facial lines).

[0346] 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.