Therapeutic and cosmetic uses of botulinum neurotoxin serotype E

Abstract

Therapeutic and cosmetic uses of botulinum neurotoxin serotype E The present invention relates to botulinum neurotoxin serotype E (BoNT / E) for use in the treatment of disorders, wherein BoNT / E is administered to a human subject and results in maximal inhibition of neurotransmitter secretion from target cells or tissues within 13 days of administration, which decreases to greater than 25% inhibition of neurotransmitter secretion from said target cells or tissues by 14 days after administration.

Description

[Technical field]

[0001] The present invention relates to clostridial neurotoxins and their therapeutic and cosmetic uses. [Background technology]

[0002] Bacteria of the genus Clostridium produce highly potent and specific protein toxins that can harm neurons and other cells to which they are delivered. Examples of such Clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes A-G and X (see WO2018 / 009903A2), as well as those produced by C. baratii and C. butyricum.

[0003] Clostridial neurotoxins include some of the most potent toxins known. As an example, botulinum neurotoxins have a median lethal dose (LD ) in mice, depending on the serotype. 50 ) values ​​range from 0.5 to 5 ng / kg. Both tetanus toxin and botulinum toxin act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Tetanus toxin acts in the central nervous system, while botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system.

[0004] In nature, clostridial neurotoxins are synthesized as single-chain polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, located between cysteine ​​residues that provide the interchain disulfide bond. This two-chain form is the active form of the toxin. The two chains are referred to as the heavy chain (H chain) with a molecular weight of approximately 100 kDa and the light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain is linked to an N-terminal translocation component (H N domain) and the C-terminal targeting moiety (H C The cleavage site is located between the L chain and the translocation domain component.C Following binding of the H domain to its target neuron and internalization of the bound toxin into the cell via endosomes, N The domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0005] The non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin) (see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc.). SNARE is derived from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-sensitive factor. SNARE proteins are essential for intracellular vesicle fusion and thus secretion of molecules from cells by vesicular transport. The protease function is a zinc-dependent endopeptidase activity and shows high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cells, the non-cytotoxic proteases can inhibit cellular secretion from the target cells. The L-chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

[0006] Botulinum neurotoxins are known for their ability to cause flaccid muscle paralysis. Due to this muscle relaxant property, botulinum neurotoxins (such as BoNT / A) are used in a wide range of medical and cosmetic procedures, including the treatment of glabellar or hyperkinetic facial wrinkles, headaches, hemifacial spasms, bladder hypermotility, hyperhidrosis, nasolabial folds, cervical dystonia, blepharospasm, and spasticity.

[0007] However, BoNT / A is associated with long-term effects (e.g., 6 months or more) that may be disadvantageous in certain therapeutic or cosmetic situations. Thus, there remains a need for alternative treatments that have pharmacodynamic properties different from those of BoNT / A. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention overcomes one or more of the problems set forth above. [Means for solving the problem]

[0009] The present inventors have determined, for the first time, the pharmacodynamic properties of BoNT / E (e.g., BoNT / E lacking the complexing proteins present in native BoNT / E) when administered to a human subject. Surprisingly, the present inventors have found that BoNT / E has pharmacodynamic properties that differ from BoNT / A, including a shorter time to achieve maximal inhibition of neurotransmitter release from target cells or tissues and a shorter duration of effect (e.g., less than 150 days after administration).

[0010] Although BoNT / E has been tested in in vitro systems and non-human animal models (e.g., mice), previous studies performed with naturally complexed BoNT / E are not suitable for predicting the pharmacodynamic properties of unconjugated (e.g., recombinant) BoNT / E, and these different forms of BoNT are known to behave differently in vivo. For example, complexed and unconjugated BoNT exhibit significantly different host cell responses in human neuronal cells. Effect of the Invention

[0011] Based on the inventors' findings, the clinical utility of BoNT / E appropriate for achieving therapeutic effects in human subjects can be determined. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Thus, in one aspect, the invention provides botulinum neurotoxin serotype E (BoNT / E) for use in treating a disorder. Related aspects include methods for treating a disorder and the use of BoNT / E in the manufacture of a medicament for treating a disorder, where BoNT / E is administered to a human subject.

[0013] In one aspect, there is provided a botulinum neurotoxin serotype E (BoNT / E) for use in treating a disorder, wherein the BoNT / E is administered to a human subject and results in maximal inhibition of neurotransmitter secretion from a target cell or tissue within 13 days after administration, which is reduced to greater than 25% inhibition of neurotransmitter secretion from the target cell or tissue by 14 days after administration.

[0014] Another aspect provides a method for treating a disorder, the method comprising administering BoNT / E to a human subject, wherein administering BoNT / E results in maximal inhibition of neurotransmitter secretion from a target cell or tissue within 13 days after administration, which decreases to greater than 25% inhibition of neurotransmitter secretion from the target cell or tissue on the 14th day after administration.

[0015] Another aspect provides a cosmetic method comprising administering BoNT / E to a human subject, wherein administering BoNT / E results in maximal inhibition of neurotransmitter secretion from target cells or tissues within 13 days after administration, which decreases to greater than 25% inhibition of neurotransmitter secretion from target cells or tissues on the 14th day after administration.

[0016] Yet another aspect provides the use of BoNT / E in the manufacture of a medicament for treating a disorder, wherein BoNT / E is administered to a human subject and results in maximal inhibition of neurotransmitter secretion from a target cell or tissue within 13 days after administration, which is reduced to greater than 25% inhibition of neurotransmitter secretion from the target cell or tissue on the 14th day after administration.

[0017] BoNT / E is preferably in an uncomplexed form (i.e., does not contain the complexing proteins present in native BoNT / E). Examples of such complexing proteins include neurotoxin-associated proteins (NAPs) and non-toxic non-hemagglutinin components (NTNHs).

[0018] One advantage of the uncomplexed form of BoNT / E is that the total weight by protein content is provided by the neurotoxin (BoNT / E) itself. Thus, for example, administration of 5 ng of uncomplexed BoNT / E typically corresponds to administration of 5 ng of BoNT / E neurotoxin. On the other hand, administration of 5 ng of complexed BoNT / E (preferably, the total weight by protein content is determined by absorbance at A280 or, for example, by Bradford assay) includes the complexed protein(s) and results in a lower dose of BoNT / E (i.e., less than 5 ng).

[0019] An uncomplexed form of BoNT / E (as described above) typically contains less than 50 picograms (pg) of complexed protein per 100 nanograms (ng) of BoNT / E protein, e.g., less than 20 pg of complexed protein per 100 ng of BoNT / E protein. In one embodiment, an uncomplexed form of BoNT / E contains less than 10 pg of complexed protein per 100 ng of BoNT / E protein, e.g., less than 5 pg of complexed protein per 100 ng of BoNT / E protein.

[0020] The BoNT / E of the present invention is preferably recombinant BoNT / E (and thus does not contain complex proteins).

[0021] The term "treatment" or "treating" as used herein includes preventative treatment (e.g., preventing the onset of a disease) and corrective treatment (treatment of a subject already suffering from a disease). Preferably, "treatment" or "treating" as used herein means corrective treatment. The term "treatment" or "treating" includes treating both a disease and its symptoms. In some embodiments, "treatment" or "treating" refers to a symptom of a disease.

[0022] Thus, BoNT / E can be administered to a human subject in a therapeutically or prophylactically effective amount.

[0023] A "therapeutically effective amount" is any amount of BoNT / E that, when administered alone or in combination to a human subject for treating a disease (or a symptom thereof), is sufficient to effect treatment for such disease or symptom thereof.

[0024] A "prophylactically effective amount" is any amount of BoNT / E that, when administered alone or in combination to a human subject, inhibits or delays the onset or recurrence of a disease (or symptoms thereof). In some embodiments, a prophylactically effective amount prevents the onset or recurrence of a disease entirely. "Inhibiting" onset means reducing the likelihood of a disease onset (or symptoms thereof) or preventing onset entirely.

[0025] The term "inhibition of neurotransmitter release from a tissue" may refer to inhibition of neurotransmitter release from nerves within said tissue.

[0026] The present invention further provides a cosmetic method comprising administering BoNT / E to a human subject.

[0027] BoNT / E may be administered to a human subject in any manner suitable to achieve the desired therapeutic effect. In one embodiment, BoNT / E is administered parenterally. Such administration may be via local injection. In one embodiment, BoNT / E is administered by subcutaneous, intradermal, and / or intramuscular injection. Preferably, BoNT / E is administered intramuscularly, e.g., by intramuscular injection.

[0028] The total dose of BoNT / E administered to a human subject will typically be 0.03 ng or more and typically 50 ng or less. For example, in some embodiments, the total dose of BoNT / E administered will be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, or 50 ng.

[0029] The term "total dose" refers to the absolute amount (expressed in grams or nanograms) of BoNT / E administered to a patient per treatment regimen. For example, a total dose of BoNT / E of 5 ng means that 5 ng of BoNT / E is administered to a human patient to complete the treatment regimen for that patient.

[0030] The total dose may be administered by one or more administration steps forming part of the same treatment regimen, for example, a total dose of 5 ng may be administered by a first administration of 2 ng BoNT / E and a second administration of 3 ng BoNT / E.

[0031] Preferably, the total dose is administered in a single administration step.

[0032] The total dose of BoNT / E administered is typically less than 50 ng, for example, less than 40, 30, 20, or 10 ng.

[0033] One advantage of the present invention is that it provides a rapid onset of action (e.g., inhibition of neurotransmitter release), which is highly desirable for many clinical and cosmetic indications. Another advantage of the present invention is that local diffusion of BoNT / E to adjacent muscles is substantially avoided (i.e., no noticeable effects are observed).

[0034] Thus, according to the invention, BoNT / E, when administered (e.g., at a dose), results in at least 15% inhibition of neurotransmitter secretion from a target cell or tissue within 1 day, preferably 15 hours, after administration. For example, BoNT / E, when administered (e.g., at a dose), results in at least 20% inhibition of neurotransmitter secretion from a target cell or tissue within 1 day, preferably 15 hours, after administration.

[0035] In one embodiment, BoNT / E, when administered, results in greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80% inhibition of neurotransmitter secretion from a target cell or tissue 14 days after administration (e.g., at a dose). For example, BoNT / E, when administered, preferably results in greater than 25% inhibition of neurotransmitter secretion from a target cell or tissue 14 days after administration (e.g., at a dose). BoNT / E, when administered, results in greater than 25% inhibition of neurotransmitter secretion from a target cell or tissue 14-49 days after administration (e.g., at a dose).

[0036] In another embodiment, BoNT / E, when administered (e.g., at a dose), results in 40-95% inhibition of neurotransmitter secretion from a target cell or tissue within 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, or 7 days after administration. For example, BoNT / E, when administered (e.g., at a dose), results in 40-60% inhibition of neurotransmitter secretion from a target cell or tissue within 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, or 7 days after administration.

[0037] In one embodiment, BoNT / E, when administered, (e.g., at a dose) results in 40-95% inhibition of neurotransmitter secretion from a target cell or tissue within 6 days after administration. For example, BoNT / E, when administered, (e.g., at a dose) results in 40-60% inhibition of neurotransmitter secretion from a target cell or tissue within 6 days after administration.

[0038] In another embodiment, BoNT / E is administered to a human subject and results in at least 40% inhibition of neurotransmitter secretion from a target cell or tissue (e.g., at a dose) within 13 days, preferably within 6 days, of administration. For example, BoNT / E is administered to a human subject and results in at least 50% inhibition of neurotransmitter secretion from a target cell or tissue (e.g., at a dose) within 13 days, preferably within 6 days, of administration.

[0039] In one embodiment, BoNT / E, when administered, provides maximal inhibition of neurotransmitter secretion from a target cell or tissue (e.g., at a certain dose) within 15 days, preferably within 10 days, and more preferably within 7 days after administration.

[0040] Another advantage of the present invention is that it provides a rapid offset in action (i.e., offset after maximal inhibition of neurotransmitter secretion from target cells or tissues) compared to BoNT / A, which is highly desirable for many clinical indications.

[0041] According to the present invention, BoNT / E, when administered, (e.g., at a certain dose) At least 40% inhibition of neurotransmitter secretion from target cells or tissues within 13 days (preferably within 12 days or within 10 days) after administration; and resulting in a 15% or less reduction in inhibition of neurotransmitter secretion from target cells or tissues more than 21 days but less than 100 days after administration.

[0042] For example, BoNT / E, when administered (e.g., at a certain dose) At least 40% inhibition of neurotransmitter secretion from target cells or tissues within 13 days after administration; and a reduction in inhibition of neurotransmitter secretion from target cells or tissues to 15% or less more than 21 days and less than 80 days after administration.

[0043] In one embodiment, BoNT / E, when administered, (e.g., at a certain dose) At least 40% inhibition of neurotransmitter secretion from target cells or tissues within 13 days after administration; and a reduction in inhibition of neurotransmitter secretion from target cells or tissues to 15% or less more than 40 days and less than 100 days after administration.

[0044] For example, BoNT / E may be administered to a human subject (e.g., at a dose) At least 40% inhibition of neurotransmitter secretion from target cells or tissues within 13 days (preferably within 12 days or within 10 days) after administration; and a reduction in inhibition of neurotransmitter secretion from target cells or tissues to 15% or less more than 40 days and less than 80 days after administration.

[0045] Inhibition of neurotransmitter secretion from target cells or tissues (preferably target tissues) can be assessed by routine techniques known to those skilled in the art. In one embodiment, the "percentage of inhibition of neurotransmitter secretion" as used herein corresponds to the percentage of inhibition of muscle action potential (e.g., compound muscle action potential, CMAP). Inhibition of muscle action potential can be assessed using standard techniques. For example, the action potential of stimulated muscle can be measured before and after BoNT / E administration via electrodes to determine the percentage of inhibition. For example, electrodes can be used in electrophysiological studies to measure the action potential (produced by neurotransmitter secretion) in tissue to determine the percentage of inhibition of the action potential (corresponding to the percentage of inhibition of neurotransmitter release).

[0046] Thus, in one embodiment, the percentage inhibition of neurotransmitter secretion from a target cell or tissue is measured using electrophysiology. For example, the percentage inhibition of action potentials (measured using electrophysiology) may be measured to provide a readout of the percentage inhibition of neurotransmitter secretion.

[0047] The main advantage of using electrophysiology to measure the percentage inhibition of neurotransmitter secretion is that it provides a standardized approach that provides objective results. This is in contrast to subjective methods that measure the percentage inhibition of neurotransmitter secretion, such as by estimating the percentage inhibition based on manual / human (e.g., physician) assessment of the response to treatment, such as manual assessment of muscle spasm or facial wrinkle levels after treatment. The results of such subjective methods are subject to a high degree of variability and do not have the precision and reliability of objective methods.

[0048] The "percentage of inhibition of neurotransmitter secretion" may correspond to the percentage of inhibition measured in superficial tissues that are particularly accessible for electrophysiological studies, such as the extensor digitorum brevis (EDB). Measuring the percentage of inhibition of neurotransmitter secretion in such superficial tissues may provide a more accurate / reliable readout (compared to measurements in less superficial tissues), which has the advantage that it may be extrapolated to the percentage of inhibition of neurotransmitter secretion in alternative tissues (targeted for therapeutic or cosmetic purposes).

[0049] Assessment of the "percentage of inhibition of neurotransmitter secretion" is demonstrated by reference to the accompanying Examples and Materials and Methods, and may be assessed using the methodology described therein (e.g., the pharmacodynamic models described therein).

[0050] In one embodiment, the neurotransmitter is acetylcholine.

[0051] The inventors have found that administration of BoNT / E provides (e.g., at a dose) a rapid onset of inhibition of neurotransmitter secretion followed by a gradual (substantially linear) recovery of neurotransmitter secretion (following maximal inhibition). Such a gradual recovery may have the advantage of being dose independent, and has been demonstrated by the inventors at doses ranging from 0.04 ng to approximately 4.0 ng.

[0052] Thus, the present invention has the advantage of providing a total dose of BoNT / E that results in a predictable, substantially linear decrease in "percent inhibition of neurotransmitter secretion" (following maximal inhibition) in a human subject, such that a subject's response to treatment may be predicted up to 100 days after administration (see FIG. 12). For example, if BoNT / E suppresses symptoms of a disorder, a prediction of the time at which symptoms may begin to recur may be made, along with a prediction of the severity of the symptoms that will recur at a particular time point.

[0053] Thus, in one embodiment, BoNT / E, when administered, produces a maximal inhibition (eg, at a given dose) followed by a substantially linear decrease in the "percent inhibition of neurotransmitter secretion."

[0054] The term "substantially linear" means that the profile (plot) of the "percentage inhibition of neurotransmitter secretion" values ​​can be fitted with a linear curve (Microsoft Excel 2010, Microsoft) having an R-squared value of greater than 0.8. In one embodiment, the R-squared value is greater than 0.85, greater than 0.9, or greater than 0.95. Preferably, the R-squared value can be greater than 0.9. More preferably, the R-squared value can be greater than 0.95.

[0055] In one embodiment, BoNT / E, when administered, results in a substantially linear decrease in "percent inhibition of neurotransmitter secretion" after maximum inhibition (e.g., at a certain dose), which substantially linear decrease extends from 10 to 56 days, 10 to 42 days, and / or 10 to 21 days after administration.

[0056] Preferably, the profiles over 10 to 56 days, 10 to 42 days, and / or 10 to 21 days post-administration represent the "wear-off" of the BoNT / E effect (inhibition of neurotransmitter secretion) following maximal inhibition of neurotransmitter secretion.

[0057] In one embodiment, BoNT / E, when administered, results in maximal inhibition of neurotransmitter secretion from target cells or tissues within 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, or 6 days (preferably within 6 days) of administration (e.g., at a given dose), and thereafter results in a 1-10% decrease per day in the percentage inhibition of neurotransmitter secretion from target cells or tissues.

[0058] The maximal inhibition (percentage of inhibition of neurotransmitter secretion) can be 40% or more, 60% or more, 75% or more, or 80% or more (e.g., 85% or more). Preferably, the maximal inhibition is greater than 80%.

[0059] In one embodiment, the maximal inhibition (percentage of inhibition of neurotransmitter secretion) is 95% or greater.

[0060] The percentage inhibition of neurotransmitter secretion from target cells or tissues usually decreases by about 2-10% per day after maximal inhibition.

[0061] For example, the percentage inhibition of neurotransmitter secretion from the target cell or tissue is decreased by about 1-4%, about 4-6%, about 6-8%, or about 8-10% per day after maximum inhibition. Preferably, the percentage inhibition of neurotransmitter secretion from the target cell or tissue is decreased by about 1-4% per day after maximum inhibition.

[0062] The percentage inhibition of neurotransmitter secretion from target cells or tissues typically decreases by about 1-4% each day on the 10-21 day, 6-42 day, 10-42 day, 10-49 day, and / or 10-56 day post-administration.

[0063] For convenience, the reduction in percentage inhibition of neurotransmitter secretion may be calculated as the average reduction over the time frame of interest, e.g., 10-24 days, 6-42 days, 10-42 days, 10-49 days, and / or 10-56 days after administration.

[0064] The percentage inhibition of neurotransmitter secretion from a target cell or tissue can decrease by about 1-30% per week, week after maximum inhibition, for example, the percentage inhibition of neurotransmitter secretion from a target cell or tissue decreases by about 1-28%, 2-28%, about 2-22%, about 4-16%, about 9-18%, about 14-16%, about 9-12% per week, week after maximum inhibition.

[0065] In one embodiment, the percentage inhibition of neurotransmitter secretion from target cells or tissues is decreased by about 9-17% each week for weeks 1-3, 1-6, and / or 2-6 after administration.

[0066] For convenience, the reduction in percentage inhibition of neurotransmitter secretion may be calculated as the average reduction over the time frame of interest, for example, 1-6 weeks, 2-6 weeks, and / or 1-3 weeks after administration.

[0067] The maximal inhibition (percentage of inhibition of neurotransmitter secretion) can be 40% or more, 60% or more, 75% or more, or 80% or more (e.g., 85% or more). Preferably, the maximal inhibition is greater than 80%.

[0068] In one embodiment, the maximal inhibition (percentage of inhibition of neurotransmitter secretion) is greater than 95%.

[0069] The percentage inhibition of neurotransmitter secretion may plateau after a substantially linear decrease in the "percentage inhibition of neurotransmitter secretion."

[0070] In one embodiment, BoNT / E, when administered (e.g., at a dose), provides about 10% to 40% (preferably about 15% to 40%) inhibition of neurotransmitter secretion from target cells or tissues, greater than 49 days to less than 100 days, or greater than 49 days to less than 90 days after administration. In one embodiment, BoNT / E, when administered (e.g., at a dose), provides about 10% to 40% (preferably about 15% to 40%) inhibition of neurotransmitter secretion from target cells or tissues, greater than 42 days to less than 100 days, or greater than 42 days to less than 90 days after administration.

[0071] When used, the total dose of BoNT / E administered is generally less than 10 ng, for example, in the range of 0.04 to 5 ng. In one embodiment, BoNT / E is administered at a dose of 0.5 to 5 ng (e.g., 0.9 ng or 3.6 ng). In another embodiment, BoNT / E is administered at a dose of 0.1 to 0.5 ng (e.g., 0.2 ng). In one embodiment, BoNT / E is administered at a dose of 1 to 5 ng (e.g., 3.6 ng). In another embodiment, BoNT / E is administered at a dose of 0.5 to 2 ng (e.g., 0.9 ng).

[0072] In one embodiment, BoNT / E, when administered at a dose of 0.5 to 5 ng (e.g., 0.9 ng or 3.6 ng), results in: Inhibition of at least 85% of neurotransmitter secretion from target cells or tissues 7 days after administration; and / or At least 80% inhibition of neurotransmitter secretion from target cells or tissues 14 days after administration, and / or At least 70% inhibition of neurotransmitter secretion from target cells or tissues 21 days after administration, and / or At least 50% inhibition of neurotransmitter secretion from target cells or tissues 28 days after administration, and / or At least 40% inhibition of neurotransmitter secretion from target cells or tissues 35 days after administration, and / or At least 30% inhibition of neurotransmitter secretion from target cells or tissues 42 days after administration, and / or At least 25% inhibition of neurotransmitter secretion from target cells or tissues 49 days after administration.

[0073] For example, BoNT / E, when administered at a dose of 0.5 to 5 ng (e.g., 0.9 ng or 3.6 ng), results in: At least 15% inhibition of neurotransmitter secretion from target cells or tissues 56 days after administration, and / or Less than 15% inhibition of neurotransmitter secretion from target cells or tissues 63 days after administration.

[0074] In another embodiment, BoNT / E, when administered at a dose of 0.1 to 0.5 ng (e.g., 0.2 ng), results in: Inhibition of neurotransmitter secretion from target cells or tissues by at least 65% 7 days after administration; and / or Inhibition of neurotransmitter secretion from target cells or tissues by at least 65% 14 days after administration; and / or At least 50% inhibition of neurotransmitter secretion from target cells or tissues 21 days after administration, and / or At least 30% inhibition of neurotransmitter secretion from target cells or tissues 28 days after administration, and / or At least 30% inhibition of neurotransmitter secretion from target cells or tissues 35 days after administration, and / or Less than 15% inhibition of neurotransmitter secretion from target cells or tissues 42 days after administration.

[0075] In one embodiment, BoNT / E, when administered at a dose of 1 to 5 ng (e.g., 3.6 ng), results in: Inhibition of at least 85% of neurotransmitter secretion from target cells or tissues within about more than 3 days and up to 13 days after administration; and / or Inhibition of about 80% to 90% (preferably about 84% to 86%) of neurotransmitter secretion from target cells or tissues more than 17 days but less than 19 days after administration; and / or Inhibition of about 70% to 80% (preferably about 74% to 76%) of neurotransmitter secretion from target cells or tissues more than 20 days but less than 22 days after administration; and / or Inhibition of about 55% to 65% (preferably about 60% to 64%) of neurotransmitter secretion from target cells or tissues more than 23 days but less than 26 days after administration; and / or Inhibition of about 50% to 60% (preferably about 54% to 56%) of neurotransmitter secretion from target cells or tissues more than 27 days but less than 29 days after administration; and / or Inhibition of about 35% to 45% (preferably about 43% to 44%) of neurotransmitter secretion from target cells or tissues more than 34 days but less than 36 days after administration; and / or Inhibition of about 30% to 40% (preferably about 34% to 36%) of neurotransmitter secretion from target cells or tissues more than 41 days but less than 43 days after administration; and / or Inhibition of about 25% to 35% (preferably about 29% to 31%) of neurotransmitter secretion from target cells or tissues more than 48 days but less than 50 days after administration; and / or Inhibition of about 10% to 20% (preferably about 15% to 17%) of neurotransmitter secretion from target cells or tissues more than 55 days but less than 57 days after administration; and / or Inhibition of about 7% to 17% (preferably about 13% to 15%) of neurotransmitter secretion from target cells or tissues more than 62 days but less than 64 days after administration; and / or Less than 15% inhibition of neurotransmitter secretion from target cells or tissues more than 75 days after administration.

[0076] In another embodiment, BoNT / E, when administered at a dose of 0.5 to 2 ng (e.g., 0.9 ng), results in: Inhibition of at least 85% of neurotransmitter secretion from target cells or tissues more than 3 days but not more than 13 days after administration; and / or Inhibition of about 70% to 80% (preferably about 77% to 79%) of neurotransmitter secretion from target cells or tissues more than 17 days but less than 19 days after administration; and / or Inhibition of about 65% to 75% (preferably about 71% to 73%) of neurotransmitter secretion from target cells or tissues more than 20 days but less than 22 days after administration; and / or Inhibition of about 60% to 70% (preferably about 65% to 67%) of neurotransmitter secretion from target cells or tissues more than 23 days but less than 26 days after administration; and / or Inhibition of about 55% to 65% (preferably about 58% to 60%) of neurotransmitter secretion from target cells or tissues more than 27 days but less than 29 days after administration; and / or Inhibition of about 45% to 55% (preferably about 50% to 52%) of neurotransmitter secretion from target cells or tissues more than 34 days but less than 36 days after administration; and / or Inhibition of about 30% to 40% (preferably about 34% to 37%) of neurotransmitter secretion from target cells or tissues more than 41 days but less than 43 days after administration; and / or Inhibition of about 30% to 40% (preferably about 33% to 35%) of neurotransmitter secretion from target cells or tissues more than 48 days but less than 50 days after administration; and / or Inhibition of about 30% to 40% (preferably about 35% to 37%) of neurotransmitter secretion from target cells or tissues more than 55 days but less than 57 days after administration; and / or Inhibition of about 20% to 30% (preferably about 22% to 27%) of neurotransmitter secretion from target cells or tissues more than 62 days but less than 64 days after administration; and / or 15% or less inhibition of neurotransmitter secretion from target cells or tissues greater than 75 days (e.g., greater than 85 days) after administration.

[0077] In one embodiment, BoNT / E, when administered at a dose of 0.1 to 0.5 ng (e.g., 0.2 ng), results in: Inhibition of at least 70% of neurotransmitter secretion from target cells or tissues more than 3 days but not more than 13 days after administration; and / or Inhibition of about 47% to 57% (preferably about 52 to 54%) of neurotransmitter secretion from target cells or tissues more than 17 days but less than 19 days after administration; and / or Inhibition of about 45% to 55% (preferably about 50% to 52%) of neurotransmitter secretion from target cells or tissues more than 20 days but less than 22 days after administration; and / or Inhibition of about 25% to 35% (preferably about 29% to 31%) of neurotransmitter secretion from target cells or tissues more than 23 days but less than 26 days after administration; and / or Inhibition of about 25% to 35% (preferably about 31% to 33%) of neurotransmitter secretion from target cells or tissues more than 27 days but less than 29 days after administration; and / or Inhibition of about 25% to 35% (preferably about 30% to 33%) of neurotransmitter secretion from target cells or tissues more than 34 days but less than 36 days after administration; and / or Inhibition of about 1% to 10% (preferably about 4% to 6%) of neurotransmitter secretion from target cells or tissues more than 41 days but less than 43 days after administration; and / or Less than or equal to 15% inhibition of neurotransmitter secretion from target cells or tissues more than 48 days after administration.

[0078] In some embodiments, the total dose of BoNT / E administered is preferably less than 0.2 ng, for example 0.04-0.15 ng (or 0.01-0.04 ng). Advantageously, the inventors have found that by administering lower doses of BoNT / E (less than 0.2 ng), at least 40% inhibition of neurotransmitter secretion from target cells or tissues is achieved. Such low doses are associated with good maximal inhibition of neurotransmitter secretion (e.g., at least 40% or 50% inhibition), a significantly shorter duration of effect, and / or a similar time to maximal effect. This latter point is surprising and is the opposite of BoNTs containing complex proteins (e.g., BoNT / A containing complex proteins), which achieve a significantly shorter time to maximal inhibition when administered at higher doses (e.g., doses of 0.2 ng or more). For example, the inventors found that as little as 0.04 ng (40 pg) of BoNT-E (e.g., recombinant BoNT-E) produced the same amplitude of effect (1-7 days after administration) as 20 U of BoNT-A (Dysport®) (see Example 4). In particular, 20 U Dysport = 108 pg neurotoxin (5.35-5.38 pg neurotoxin / U, typically 5.35 pg neurotoxin / U). This was particularly surprising to the inventors.

[0079] In one embodiment, BoNT / E, when administered at a dose of 0.04 to 0.15 ng (e.g., 0.04 ng), results in: At least 40% inhibition of neurotransmitter secretion from target cells or tissues more than 3 days but less than 13 days after administration; and / or Inhibition of about 30% to 40% (preferably about 33 to 35%) of neurotransmitter secretion from target cells or tissues more than 17 days but less than 19 days after administration; and / or Inhibition of about 7% to 17% (preferably about 12% to 14%) of neurotransmitter secretion from target cells or tissues more than 20 days but less than 22 days after administration; and / or Inhibition of about 15% to 25% (preferably about 21% to 23%) of neurotransmitter secretion from target cells or tissues more than 23 days but less than 26 days after administration; and / or Less than or equal to 15% inhibition of neurotransmitter secretion from target cells or tissues more than 48 days after administration.

[0080] In one embodiment, the target tissue is a muscle. Examples of such muscles include the occipitalis, nasus, orbicularis oris, depressor anguli oris, platysma, sternohyoid, serratus anterior, rectus abdominis, external oblique, tensor fasciae latae, brachioradialis, iliacus, psoas major, pubic, adductor longus, sartorius, gracilis, vastus lateralis, rectus femoris, vastus medialis, quadriceps tendon, patella, gastrocnemius, soleus, tibia, peroneus longus, tibialis anterior, patellar ligament, iliotibial band, hypothenar group, These include the thenar muscles, flexor carpi ulnaris, flexor digitorum superficialis, palmaris longus, flexor carpi radialis, brachioradialis, pronator teres, brachialis, biceps brachii, triceps brachii, pectoralis major, deltoid, trapezius, sternocleidomastoid, masseter, orbicularis oculi, temporalis, galea aponeurosis, teres major, extensor digitorum major, extensor carpi ulnaris, elbow, abductor pollicis longus, plantaris, Achilles tendon, soleus, adductor magnus, gluteus maximus, gluteus medius, latissimus dorsi, and infraspinatus.

[0081] In one embodiment, the target tissue is one or more selected from the corrugator supercilii, triceps brachii, biceps brachii, brachialis, and / or brachioradialis, extensor digitorum, or combinations thereof.

[0082] In one embodiment, the target tissue is one or more selected from the flexor carpi radialis, flexor carpi ulnaris, flexor digitorum profundus, flexor digitorum superficialis, flexor pollicis longus, adductor pollicis longus, brachialis, brachioradialis, biceps brachii, pronator teres, triceps brachii, pectoralis major, subscapularis, latissimus dorsi, or combinations thereof (preferably, BoNT / E is used to treat upper limb spasticity).

[0083] In one embodiment, the target tissue is one or more selected from flexor carpi radialis, flexor carpi ulnaris, or a combination thereof. In one embodiment, the target tissue is one or more selected from flexor digitorum profundus, flexor digitorum superficialis, flexor pollicis longus, adductor pollicis, or a combination thereof. In one embodiment, the target tissue is one or more selected from brachialis, brachioradialis, biceps brachii, pronator teres, or a combination thereof. In one embodiment, the target tissue is one or more selected from triceps brachii, pectoralis major, subscapularis, latissimus dorsi, or a combination thereof.

[0084] In one embodiment, the target tissue is one or more selected from the soleus muscle, gastrocnemius muscle (e.g., medial or lateral head), tibialis posterior muscle, flexor digitorum longus, flexor digitorum brevis, flexor hallucis longus, flexor hallucis brevis, or combinations thereof (preferably, BoNT / E is used to treat lower limb spasticity).

[0085] In one embodiment, the target tissue is the soleus muscle. In one embodiment, the target tissue is the gastrocnemius muscle (e.g., medial head or lateral head). In one embodiment, the target tissue is the tibialis posterior muscle, flexor digitorum longus, flexor digitorum brevis, flexor hallucis longus, flexor hallucis brevis, or a combination thereof.

[0086] In one embodiment, the target tissue is one or more selected from the corrugator supercilii, procerus, orbicularis oculi, frontalis, or combinations thereof (preferably, BoNT / E is used to treat upper facial lines).

[0087] In one embodiment, the target tissues are the corrugator supercilii and one or more selected from the procerus, orbicularis oculi, frontalis, or combinations thereof. In one embodiment, the target tissues are the procerus and one or more selected from the corrugator supercilii, orbicularis oculi, frontalis, or combinations thereof. In one embodiment, the target tissues are the orbicularis oculi and one or more selected from the corrugator supercilii, orbicularis oculi, frontalis, or combinations thereof. In one embodiment, the target tissues are the frontalis and one or more selected from the corrugator supercilii, orbicularis oculi, frontalis, or combinations thereof.

[0088] In one embodiment, the target cell is a motor neuron or a sensory neuron. Preferably, the target cell is a motor neuron.

[0089] Target cells (used herein synonymously with the term "nerve") include axillary nerve, phrenic nerve, spinal ganglion, spinal nerve, sympathetic ganglion chain, pudendal nerve, common palmar digital nerve, ulnar nerve, deep branch of ulnar nerve, sciatic nerve, peroneal nerve, tibial nerve, saphenous nerve, interosseous nerve, superficial peroneal nerve, intermediate dorsal cutaneous nerve, medial plantar nerve, medial dorsal cutaneous nerve, deep peroneal nerve, muscular branch of tibial nerve, infrapatellar branch of saphenous nerve, common peroneal nerve, muscular branch of femoral nerve, anterior cutaneous branch of femoral nerve, muscular branch of sciatic nerve, femoral nerve, ilioinguinal nerve, filum terminale nerve, iliohypogastric nerve, obturator nerve, radial nerve, subcostal nerve, intercostal nerve, dorsal branch of intercostal nerve, medial cutaneous branch of intercostal nerve, musculaneous nerve, deltoid nerve, The target nerve may be the peroneal nerve, vagus nerve, brachial plexus nerve, supraclavicular nerve, facial nerve, auriculotemporal nerve, or a combination thereof. In a preferred embodiment, the target nerve is the peroneal nerve.

[0090] A BoNT / E for use in the present invention may be encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1. In one embodiment, a BoNT / E for use in the present invention may be encoded by a nucleotide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 1. Preferably, a BoNT / E for use in the present invention may be encoded by a nucleotide sequence having (or consisting of) SEQ ID NO: 1. Preferably, a BoNT / E for use in the present invention may be encoded by a nucleotide sequence having (or consisting of) a sequence (e.g., SEQ ID NO: 1) that is codon-optimized for expression in a heterologous expression system, such as an E. coli host cell.

[0091] A BoNT / E for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, a BoNT / E for use in the present invention may comprise a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3. Preferably, a BoNT / E for use in the present invention comprises (or consists of) the polypeptide sequence shown as SEQ ID NO: 2 or SEQ ID NO: 3.

[0092] Preferably, a BoNT / E for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2. Preferably, a BoNT / E for use in the present invention may comprise a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 2. More preferably, a BoNT / E for use in the present invention comprises (or consists of) the polypeptide sequence shown as SEQ ID NO: 2.

[0093] A BoNT / E for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:2, provided that the polypeptide sequence comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight, preferably all eight) of the following amino acids (numbering of the amino acid positions begins 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, and asparagine at position 1195.

[0094] In one embodiment, a BoNT / E for use in the invention may comprise a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO:2, provided that the polypeptide sequence comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight, preferably all eight) of the following amino acids (numbering of the amino acid positions begins 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, and asparagine at position 1195.

[0095] The amino acid may be a substitution (e.g., a mutation) compared to a wild-type BoNT / E polypeptide sequence (such as SEQ ID NO:3). For example: The glycine at position 177 may be an arginine to glycine substitution (R177G); The serine at position 198 may be a C198S substitution, The alanine at position 340 may be an R340A substitution; The leucine at position 773 may be an I173L substitution; The leucine at position 963 may be an F963L substitution; The glutamine at position 964 may be substituted with E964Q. The alanine at position 967 may be an R967A substitution; and / or The asparagine at position 1195 can be an insertion (eg, an insertion between G1194 and N1195 of a wild-type BoNT / E sequence, such as the polypeptide sequence of SEQ ID NO:3).

[0096] In one embodiment, the one or more amino acids include (or consist of) 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, and asparagine at position 1195.

[0097] In one embodiment, the one or more amino acids include (or consist of) glycine at position 177, alanine at position 340, leucine at position 773, leucine at position 963, glutamine at position 964, alanine at position 967, and asparagine at position 1195.

[0098] In one embodiment, the one or more amino acids include (or consist of) a glycine at position 177 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a serine at position 198, an alanine at position 340, a leucine at position 773, a leucine at position 963, a glutamine at position 964, an alanine at position 967, and an asparagine at position 1195.

[0099] In one embodiment, the one or more amino acids include (or consist of) a serine at position 198 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a glycine at position 177, an alanine at position 340, a leucine at position 773, a leucine at position 963, a glutamine at position 964, an alanine at position 967, and an asparagine at position 1195.

[0100] In one embodiment, the one or more amino acids include (or consist of) an alanine at position 340 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a glycine at position 177, a serine at position 198, a leucine at position 773, a leucine at position 963, a glutamine at position 964, an alanine at position 967, and an asparagine at position 1195.

[0101] In one embodiment, the one or more amino acids include (or consist of) a leucine at position 773 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a glycine at position 177, a serine at position 198, an alanine at position 340, a leucine at position 963, a glutamine at position 964, an alanine at position 967, and an asparagine at position 1195.

[0102] In one embodiment, the one or more amino acids include (or consist of) a leucine at position 963 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a glycine at position 177, a serine at position 198, an alanine at position 340, a leucine at position 773, a glutamine at position 964, an alanine at position 967, and an asparagine at position 1195.

[0103] In one embodiment, the one or more amino acids include (or consist of) a glutamine at position 964 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a glycine at position 177, a serine at position 198, an alanine at position 340, a leucine at position 773, a leucine at position 963, an alanine at position 967, an asparagine at position 1195.

[0104] In one embodiment, the one or more amino acids include (or consist of) an alanine at position 967 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a glycine at position 177, a serine at position 198, an alanine at position 340, a leucine at position 773, a leucine at position 963, a glutamine at position 964, and an asparagine at position 1195.

[0105] In one embodiment, the one or more amino acids include (or consist of) an asparagine at position 1195 and one or more of the following (e.g., one or more, two or more, three or more, four or more, five or more, six or more, or seven): a glycine at position 177, a serine at position 198, an alanine at position 340, a leucine at position 773, a leucine at position 963, a glutamine at position 964, and an alanine at position 967.

[0106] In one embodiment, the presence of one or more amino acids as described above provides a BoNT / E protein with improved solubility compared to a BoNT / E protein lacking the amino acid(s), which improves protein yield in a heterologous expression system, such as an E. coli expression system.

[0107] In one embodiment, the presence of the one or more amino acids as described above improves the potency of the BoNT / E protein compared to a BoNT / E protein lacking the amino acid(s), which may preferably be improved in vivo potency (more preferably improved in vivo potency in a human subject).

[0108] In one embodiment, the BoNT / E is as described in (or encoded by) the nucleotide sequence described in WO2014 / 068317A1, which is incorporated herein by reference.

[0109] BoNT / E is formed from two polypeptide chains, a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and the N-terminal translocation component (H N domain) and H chain (H C Domain and H N BoNT / E, which contains both the A domain and the L chain, is called a holotoxin.

[0110] In one embodiment, the BoNT / E used in the present invention is a holotoxin.

[0111] An example of an L chain canonical sequence includes amino acid residues 1 to 422 of BoNT / E. However, this canonical sequence should be considered a guideline and slight variations may occur depending on the serosubtype. As an example, US2007 / 0166332 (the disclosure of which is incorporated herein by reference in its entirety) cites BoNT / EL chain sequences that differ slightly from amino acid residues M1 to R422.

[0112] BoNT / EH C An example of a domain reference sequence includes amino acid residues R846 to K1252.

[0113] The light chain of BoNT / E used in the present invention can be as follows (SEQ ID NO:4): [ka]

[0114] The heavy chain of a BoNT / E for use in the present invention can be as follows (SEQ ID NO:5): [ka]

[0115] BoNT / E H C The domain is H CC Domain and H CN It contains two distinct structural features called domains. The amino acid residues involved in receptor binding are mainly H CC It is believed to be located in the BoNT / EH domain. CN An example of a domain reference sequence includes amino acid residues 846-1085.

[0116] The above sequence positions may vary slightly depending on the subtype and should be used to identify the appropriate (reference) BoNT / EH CN Another example of a domain includes amino acid residues 848-1085.

[0117] BoNT / E may be produced by C. botulinum or C. butyricum, preferably C. botulinum. In one embodiment, BoNT / E is produced in a non-clostridial cell. Alternatively (and preferably), BoNT / E may be produced in recombinant form (e.g., E. coli). Thus, in one embodiment, BoNT / E for use in the present invention is produced in a heterologous expression system, such as E. coli. In one embodiment, the E. coli cell is E. coli BLR(DE3).

[0118] Details of BoNT / E produced in a heterologous expression system (such as E. coli) are described in WO2014 / 068317A1, which is incorporated herein by reference.

[0119] The BoNT / E compositions of the present invention advantageously are substantially free of trypsin protease (used to activate single-chain polypeptides), thus preventing undesired non-specific cleavage of the BoNT / E protein.

[0120] In the BoNT / E compositions of the present invention, the protein content of the composition is provided by the actual neurotoxin (BoNT / E). Thus, for example, administration of 4 ng of a BoNT / E composition corresponds to administration of 4 ng of BoNT / E neurotoxin.

[0121] In one embodiment, where a BoNT / E composition (as described above) is substantially free of trypsin, the composition comprises less than 100 picograms (pg) of trypsin per 100 nanograms (ng) of BoNT / E protein, e.g., less than 50, 20, 10, 9, 8, 7, 6, or 5 pg of trypsin per 100 ng of BoNT / E protein. In one embodiment, a composition (as described above) comprises less than 10 pg of trypsin per 100 ng of BoNT / E protein, or less than 7 pg of trypsin per 100 ng of BoNT / E protein, or less than 5 pg of trypsin per 100 ng of BoNT / E protein. In a preferred embodiment, a composition (as described above) comprises less than 10 pg of trypsin per 100 ng of BoNT / E protein, or less than 7 pg of trypsin per 100 ng of BoNT / E protein.

[0122] Thus, in one embodiment, the phrase "substantially free of trypsin" means less than 100 pg trypsin per 100 ng BoNT / E protein, for example, less than 50, 20, 10, 9, 8, 7, 6, or 5 pg trypsin per 100 ng BoNT / E protein, preferably less than 10 pg trypsin per 100 ng BoNT / E protein, or less than 7 pg trypsin per 100 ng BoNT / E protein.

[0123] Methods for determining the concentration of trypsin in a composition are known in the art (for example, as described in WO2014 / 068317).For example, the concentration of trypsin in the composition of the present invention can be determined using sandwich ELISA (enzyme-linked immunosorbent assay).

[0124] BoNT / E is known to cleave the synaptosomal associated protein of 25 kDa (SNAP-25).

[0125] In some embodiments, the BoNT / E may be a modified BoNT / E or a derivative thereof, including but not limited to those described below. The modified BoNT / E or derivative may contain one or more amino acids that are modified compared to the native (unmodified) form of BoNT / E, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of BoNT / E. As an example, the modified BoNT / E may have a modified amino acid sequence in one or more domains compared to the native (unmodified) BoNT / E sequence. Such modifications may modify a functional aspect of BoNT / E, such as biological activity or persistence. Thus, in one embodiment, the BoNT / E is a modified BoNT / E, or a BoNT / E derivative.

[0126] Modified neurotoxins are heavy chain (modified H C The heavy chain may have one or more modifications in the amino acid sequence of the heavy chain (e.g., domain), and the modified heavy chain binds to target neurons with higher or lower affinity than native (unmodified) BoNT / E. C These modifications in the H domain alter the binding of the H domain to ganglioside receptors and / or protein receptors on target neurons. C These may include modifications of residues in the ganglioside binding site or protein (SV2 or synaptotagmin) binding site of the domain. Examples of such modified Clostridial neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0127] The modified BoNT / E can have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domains that can alter or modify the SNARE protein specificity of the modified L chain. Examples of such modified Clostridial neurotoxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference in their entireties.

[0128] The modified BoNT / E may contain one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified BoNT / E. For example, the modified BoNT / E may contain a leucine- or tyrosine-based motif that increases or decreases the biological activity and / or biological persistence of the modified neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM, where x is any amino acid. Suitable tyrosine-based motifs include Yxx-Hy, where Hy is a hydrophobic amino acid. Examples of modified clostridial neurotoxins containing leucine- and tyrosine-based motifs are described in WO2002 / 08268, the disclosure of which is incorporated herein by reference in its entirety.

[0129] The term "BoNT / E" in one embodiment encompasses hybrid and chimeric BoNT / E. A hybrid BoNT / E comprises at least a portion of a light chain from one Clostridial neurotoxin or subtype thereof and at least a portion of a heavy chain from another Clostridial neurotoxin or Clostridial neurotoxin subtype, at least one of the subtypes being a BoNT / E subtype. In one embodiment, a hybrid BoNT / E may comprise the entire light chain from one Clostridial neurotoxin subtype and a heavy chain from another Clostridial neurotoxin subtype, at least one of the subtypes being a BoNT / E subtype. In another embodiment, a chimeric Clostridial neurotoxin may comprise a portion of a heavy chain (e.g., a binding domain) from one Clostridial neurotoxin subtype together with another portion of a heavy chain from another Clostridial neurotoxin subtype, at least one of the subtypes being a BoNT / E subtype. Similarly or alternatively, a therapeutic element may comprise a light chain portion from a different BoNT / E subtype. Such hybrid or chimeric BoNT / E polypeptides are useful, for example, as a means of delivering therapeutic benefit to patients who are immune-resistant to a particular Clostridial neurotoxin subtype, who may have lower than average concentrations of receptors for a particular Clostridial neurotoxin heavy chain binding domain, or who may have protease-resistant variants of membrane or vesicle toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric Clostridial neurotoxins are described in US 8,071,110, the disclosure of which is incorporated herein by reference in its entirety. Thus, in one embodiment, the BoNT / E of the present invention is a hybrid BoNT / E or a chimeric BoNT / E.

[0130] The present invention further encompasses BoNT / E having a non-naturally occurring protease cleavage site. In such BoNT / E polypeptides, the natural protease cleavage site (also known as the activation site, as described above) is modified or replaced with a protease cleavage site that is not naturally present in BoNT / E (i.e., an exogenous cleavage site). Such sites require an exogenous protease for cleavage, thereby allowing for improved control over the timing and location of the cleavage event. Non-naturally occurring protease cleavage sites that may be utilized in BoNT / E include the following: Enterokinase (DDDDK↓) Factor Xa (IEGR↓ / IDGR↓) TEV (Tobacco Etch Virus) (ENLYFQ↓G) Thrombin (LVPR↓GS) PreScission (LEVLFQ↓GP).

[0131] Additional protease cleavage sites include recognition sequences that are cleaved by non-cytotoxic proteases, such as the light chain of a clostridial neurotoxin. These include SNARE (SNAP-25, syntaxin, VAMP, etc.) protein recognition sequences that are cleaved by non-cytotoxic proteases, such as the light chain of a clostridial neurotoxin. Clostridial neurotoxins that contain non-natural protease cleavage sites are described in US 7,132,259, EP 1206554-B2, and US 2007 / 0166332, all of which are incorporated herein by reference in their entirety. The term protease cleavage site also includes inteins, which are self-cleaving sequences. Self-splicing reactions can be controlled, for example, by varying the concentration of reducing agent present.

[0132] The present invention further encompasses BoNT / E that includes a "destructive cleavage site." In the BoNT / E polypeptide, a non-natural protease cleavage site is incorporated into BoNT / E at a location selected such that cleavage at the site reduces or inactivates the activity of BoNT / E. The destructive protease cleavage site may be susceptible to cleavage by local proteases if BoNT / E migrates to a non-target location after administration. Suitable non-natural protease cleavage sites include those described above. Clostridial neurotoxins that include destructive cleavage sites are described in WO2010 / 094905 and WO2002 / 044199, both of which are incorporated herein by reference in their entireties.

[0133] In one embodiment, BoNT / E contains a natural cleavage site.

[0134] In one embodiment, BoNT / E does not contain an E3 ligase recognition motif.

[0135] BoNT / E, particularly its light chain component, may be PEGylated, which may aid in increasing the stability, e.g., duration of action, of the light chain component. PEGylation preferably involves the addition of PEG to the N-terminus of the light chain component. As an example, the N-terminus of the light chain may be extended with one or more amino acid (e.g., cysteine) residues, which may be the same or different. One or more of said amino acid residues may have a PEG molecule attached thereto (e.g., covalently attached). An example of this approach is described in WO2007 / 104567, the disclosure of which is incorporated herein by reference in its entirety.

[0136] The BoNT / E used in the present invention can be produced using recombinant nucleic acid techniques.

[0137] BoNT / E for use in the present invention may be prepared by a method comprising expressing a nucleic acid encoding BoNT / E in a suitable host cell, lysing the host cell to provide a host cell homogenate containing single-chain BoNT / E, and isolating the single-chain BoNT / E. Prior to use in the present invention, the single-chain BoNT / E is preferably proteolytically processed into the corresponding active two-chain BoNT / E by contacting the single-chain BoNT / E with a protease that hydrolyzes a peptide bond in the BoNT / E activation loop to produce a two-chain BoNT / E (e.g., in which the light and heavy chains are linked by a disulfide bond).

[0138] Preferably, the host cell provides a heterologous (e.g. non-clostridial) expression system. Preferably, the host cell is E. coli.

[0139] The BoNT / E used in the present invention is preferably in a two-chain form.

[0140] BoNT / E may be used in the present invention as part of a pharmaceutical composition. The pharmaceutical composition may include BoNT / E and a pharma- ceutically acceptable carrier, excipient, adjuvant, and / or salt. Thus, in one embodiment, BoNT / E may be administered in combination with a pharma- ceutically acceptable carrier, excipient, adjuvant, and / or salt.

[0141] BoNT / E may be formulated for parenteral administration, continuous infusion, or topical application. In one embodiment, BoNT / E is formulated for parenteral administration, such as by subcutaneous injection, intradermal injection, and / or intramuscular injection. Preferably, BoNT / E is formulated for parenteral administration, more preferably for intramuscular administration (e.g., by injection).

[0142] Compositions suitable for injection may be in the form of solutions, suspensions, or emulsions, or as a dry powder that is dissolved or suspended in a suitable solvent prior to use.

[0143] In the case of locally delivered BoNT / E, the BoNT / E can be formulated as a cream (eg, for topical application) or for subcutaneous injection.

[0144] The local delivery means may include an aerosol or other spray device (eg, a nebulizer).

[0145] Fluid dosage forms are generally prepared using BoNT / E and a pyrogen-free sterile solvent. BoNT / E can be dissolved or suspended in the solvent depending on the solvent and the concentration used. In preparing a solution, BoNT / E can be dissolved in the solvent, and the solution is made isotonic, if necessary, by adding sodium chloride, sterilized by filtration through a bacterial filter using aseptic techniques, and then filled into suitable sterile vials or ampoules and sealed. Alternatively, if the stability of the solution is sufficient, the solution in the sealed container is sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics can be dissolved in the solvent.

[0146] Dry powders, which are dissolved or suspended in a suitable solvent before use, may be prepared by filling pre-sterilized materials into sterile containers using aseptic techniques in a sterile area. Alternatively, the materials may be dissolved in suitable containers using aseptic techniques in a sterile area. The product is then freeze-dried and the containers are aseptically sealed.

[0147] Parenteral suspensions suitable for intramuscular, subcutaneous, or intradermal injection are prepared in substantially the same manner, except that the sterile ingredients are suspended in a sterile solvent instead of being dissolved, and sterilization cannot be accomplished by filtration. The ingredients may be isolated under sterile conditions or may be sterilized after isolation, for example by gamma irradiation.

[0148] It may be advantageous to include a suspending agent, such as polyvinylpyrrolidone, in the composition(s) to facilitate uniform distribution of the ingredients.

[0149] Administration according to the invention may utilize a variety of delivery techniques including microparticle encapsulation, viral delivery systems, or high pressure aerosol impaction. BoNT / E may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of the affected organ.

[0150] The dosage ranges for administering BoNT / E will be those that produce the desired therapeutic effect (e.g., those described above). Dosages can be based on CBA units as described in the Examples.

[0151] In one embodiment, the disorder of the invention is one or more of the following: conditions associated with strabismus, blepharospasm, strabismus, dystonia (e.g., spastic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g., spasmodic torticollis), cosmetic therapeutic (facial) applications in which cell / muscle incapacitation (by downregulation or inactivation of SNAREs) is beneficial, neuromuscular disorders or may be due to eye movement conditions (e.g., conjugate strabismus, vertical strabismus, lateral rectus palsy, nystagmus, thyroid dysmyopathy), writer's cramp, blepharospasm, teeth grinding, Wilson's disease, tremors, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, anismus, back spasms, muscle rigidity, tension headaches, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorders, cerebral palsy, Locality Spasticity, spastic colitis, neurogenic bladder, anismus, limb spasticity (e.g., leg spasticity or arm spasticity), tics, tremors, teeth grinding, anal fissures, achalasia, dysphagia, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g., pain due to muscle spasms), headache pain (e.g., tension headaches), glabellar lines, skin wrinkles (e.g., upper facial lines such as glabellar lines, epicanthal lines, and / or frontal lines), cancer, uterine disorders, genitourinary disorders, genitourinary neurological disorders, chronic neurogenic inflammation, and smooth muscle disorders.

[0152] In one embodiment, the disorder is lower limb spasticity. Preferably, the lower limb spasticity is in a subject aged 18 years or older.

[0153] In one embodiment, the disorder is upper limb spasticity. Preferably, the upper limb spasticity is in a subject aged 18 years or older.

[0154] The present invention further encompasses cosmetic methods that include the use of BoNT / E. For example, the cosmetic method can be for treating upper facial lines, such as glabellar, epicanthal, and / or frontal lines.

[0155] In one embodiment, the cosmetic method is for treating glabellar lines and one or more selected from the lateral canthal lines and frontal lines. In one embodiment, the cosmetic method is for treating glabellar lines and one or more selected from the lateral canthal lines and frontal lines. In one embodiment, the cosmetic method is for treating frontal lines and one or more selected from the lateral canthal lines and frontal lines.

[0156] The cosmetic method of the present invention may comprise administering (an effective amount of) BoNT / E to treat glabellar lines, lateral canthal lines, and / or frontal lines, wherein BoNT / E is administered simultaneously or sequentially to the areas where the glabellar lines, lateral canthal lines, and / or frontal lines are to be treated, such that each of the glabellar lines, lateral canthal lines, and / or frontal lines is treated (preferably simultaneously).

[0157] Preferably, the cosmetic method is for treating glabellar lines, which can be moderate or severe (e.g., as determined by a physician).

[0158] Embodiments relating to the use of BoNT / E apply equally to the therapeutic and cosmetic methods described herein, and vice versa.

[0159] In one embodiment, the human subject is male.

[0160] sequence homology

[0161] Percent identity can be determined using any of a variety of sequence alignment methods, including, but not limited to, global methods, local methods, and hybrid methods such as, for example, segmental approaches. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and applying gap penalties. Non-limiting methods include, for example, CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994)) and iterative refinement (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992)), Gibbs sampling (see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993)), and Align-M (see, e.g., Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004)).

[0162] Thus, percent sequence identity is determined by conventional methods, see, e.g., Altschul et al., Bull. Math. Bio. 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, the two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum62" scoring matrix of Henikoff and Henikoff (supra), with amino acids designated in standard one-letter code, as shown below:

[0163] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids, multiplied by 100. The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The sequence comparison and the determination of percent identity between two or more sequences can be performed using certain mathematical algorithms, such as BLAST, which are well known to those skilled in the art.

[0164]

number

[0165] The percent identity is then calculated as follows:

number

[0166] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. Such changes are preferably minor in nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, generally small deletions of from one to about 30 amino acids, and small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0167] Conservative Amino Acid Substitutions

[0168] Basicity: Arginine lysine Histidine Acidic: Glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Ballin Aromatic: Phenylalanine Tryptophan Tyrosine Small size: glycine Alanine Serine Threonine Methionine

[0169] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues in the polypeptides. The polypeptides of the invention may include non-naturally occurring amino acid residues.

[0170] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxyethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be utilized in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system that includes E. coli S30 extracts and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993, and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993. In the second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired unnatural amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-naturally occurring amino acids are incorporated into polypeptides in place of their natural counterparts. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0171] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0172] Essential amino acids in the polypeptides of the invention can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structures, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutation of amino acids at putative contact sites. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identification of essential amino acids can also be inferred from analysis of homologies with related components (e.g., translocation or protease components) of the polypeptides of the invention.

[0173] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods in which two or more positions within a polypeptide are randomized simultaneously, functional polypeptides are selected, and then the mutagenized polypeptides are sequenced to determine the range of substitutions allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication No. WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0174] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods in which two or more positions within a polypeptide are randomized simultaneously, functional polypeptides are selected, and then the mutagenized polypeptides are sequenced to determine the range of substitutions allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication No. WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0175] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this specification belongs. In accordance with common textbooks and dictionaries. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide a common dictionary for those of ordinary skill in the art for many of the terms used in this disclosure.

[0176] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0177] The headings provided herein do not limit the various aspects or embodiments of the disclosure.

[0178] In the present specification, amino acids are designated by their names, three-letter abbreviations, or one-letter abbreviations. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by multiple nucleotide sequences.

[0179] Other definitions of terms may appear throughout the specification. Before describing example embodiments in more detail, it is to be understood that the disclosure is not limited to the particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the disclosure is defined only by the appended claims.

[0180] When a range of values ​​is presented, each intervening value between the upper and lower limits of that range is also understood to be specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly indicates otherwise. Smaller ranges between any stated or intervening value in a stated range and any other stated or intervening value in the stated range are included in the disclosure. The upper and lower limits of such smaller ranges may be independently included or excluded in the range, and each range where one or both of the upper and lower limits are included or neither are included in the smaller range is also included in the disclosure, subject to any specifically excluded limits in the stated range. When a stated range includes one or both of the limits, ranges excluding one or both of the included limits are also included in the disclosure.

[0181] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a botulinum neurotoxin" includes a plurality of such candidate agents, reference to "the botulinum neurotoxin" includes reference to one or more botulinum neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0182] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0183] Embodiments of the present invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0184] [Figure 1] FIG. 1 shows (A) the location of the extensor digitorum brevis muscle and (B) a depiction of the recorded action potentials. [Diagram 2] FIG. 2 shows the procedure and results of dose escalation of BoNT / E. [Diagram 3] FIG. 3 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over a 12-week period following administration of different doses of rBoNT / E compared to placebo (mean±SEM). [Figure 4] FIG. 4 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over 26 weeks after administration of different doses of Dysport® compared to placebo (mean±SEM). [Diagram 5] FIG. 5 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over 26 weeks following administration of different doses of rBoNT / E compared to placebo (mean±SEM). [Figure 6] FIG. 6 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over a 7-day period following administration of different doses of rBoNT / E compared to placebo (mean±SEM). [Figure 7] FIG. 7 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over a 7-day period following administration of different doses of Dysport® compared to placebo (mean±SEM). [Figure 8] FIG. 8 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over 26 weeks following administration of 0.04 ng rBoNT / E or 20 U Dysport® (mean±SEM). [Figure 9] FIG. 9 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over a 7-day period following administration of 0.04 ng rBoNT / E or 20 U Dysport® (mean±SEM). [Figure 10] FIG. 10 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over a 26-week period following administration of 0.2 ng rBoNT / E or 40 U Dysport® (mean±SEM). [Figure 11]FIG. 11 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over a 7-day period following administration of 0.2 ng rBoNT / E or 40 U Dysport® (mean±SEM). [Figure 12] Figure 12 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over 10-21 days after administration of 0.04 ng of rBoNT / E (mean ± SEM). A linear curve with an R-squared value of 0.9642 and a slope of y = 3.5531x + 9.9523 was fitted to the CMAP values ​​(Microsoft Excel). The CMAP values ​​correspond to the values ​​shown for this dose in Figure 3. [Figure 13] Figure 13 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over 10-42 days after administration of 0.9 ng rBoNT / E (mean ± SEM). A linear curve with an R-squared value of 0.9926 and a slope of y = 1.6167x - 4.8227 was fitted to the CMAP values ​​(Microsoft Excel). The CMAP values ​​correspond to the values ​​shown for this dose in Figure 3. [Figure 14] Figure 14 shows the percentage of baseline compound muscle action potential (CMAP) of stimulated extensor digitorum brevis muscle over days 10-56 after administration of 3.6 ng of rBoNT / E (mean ± SEM). A linear curve with an R-squared value of 0.9736 and a slope of y=1.7325x-9.3108 was fitted to the CMAP values ​​(Microsoft Excel). The CMAP values ​​correspond to the values ​​shown for this dose in Figure 3.

[0185] Sequence Listing

[0186] Where the first Met amino acid residue or the corresponding first codon is shown in any of the following SEQ ID NOs, that residue / codon can be optional.

[0187] SEQ ID NO:1 (nucleotide sequence of BoNT / E) 1 atgccaaaaa ttaatagtttt taattataat gatcctgtta atgatagaac attttatat 61 attack gcggttgtca agaattttat aaatcattta atattatgaa aaatattgg 121 atattccag agagaatgt aattggtaca accccaag atttcatcc gcctactca 181 ttaaaaatg gagatagtag ttattatgac cctattatt tacaagtga tgagaaag 241 gatagatttt taaaaatagt cacaaaaata tttaatagaa taataata tctttcagga 301 gggatttta taggaact gtcaaagct aatccatatt taggaatga taatactcca 361 gatacaat tccatattgg tgatgcatca gcagttgaga ttaattctc aaatggtagc 421 ciegacatac tattacta tgttatta atgggagcag agcctgattt atttgaact 481 aacagttcca atatttctct aagaaataat tatatgcca gcaatcacgg ttttggatca 541 atagctatag taacattctc acctgaatat tctttagat ttaatgata tagtagaat 601 gatttattc aagatcctgc tcttacatta atgcatgaat taatacattc attacatgga 661 ctatatgggg ctaagggat tactacaaag tatactata cacaaaaaaatccccta 721 atachaata tagaggtac aaatattgaa gatttcttta cttttggagg tactgattta 781 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc tgattataaa 841 aaatagcgt ctaaacttag caagtacaa gtactaatc cactacttaa tccttataaa 901 gatgttttg aagcaagta tggattagat aagatgcta gcggaattta ttcggtaaat 961 aaaaaaaat ttaatgat ttttaaaaaa tattacagct tacggaatt tgatttagca 1021 actaaatttc aagttaaatg taggcaact tatattggac agtataata cttcaaactt 1081 tcaacttgt taaatgattc tattaat atcagaag gctatatat aattaattta 1141 aaggtaatt tagaggaca gatgcaat ttaatccta gatttattac accattaca 1201 ggtagaggac tagtaaaaaaatcattaga tttgtaaaa atattgttc tgtaaaaggc 1261 aataggaat caatgtat cgaataat atggtgagt tattttttgt ggcttccgag 1321 atagttata atgatgaata tataatact cctaaagaa ttgacgatac agtacttca 1381 Attaatatt atgaaatga tttagatcag gttatttta attttatag tgaatcagca 1441 cctggacttt cagatgaaa atttaattta actatccaaa atgatgcttta tataccaaaa 1501 tatgattcta atgaacag tgatatagaa cacatgatg ttaatgact taatgtattt 1561 ttcttag atgcacagaa agtgccgaa ggtgaaata atgtcaatct cacctctca 1621 attgatacag cattattaga acaacctaaa atatatacat tttttcatc agaatttatt 1681 ataatgtca ataacctgt gcaagcagca ttttgtaa gctggataca acagtgtta 1741 gtagatttta ctactgaagc taaccaaaa agtactgttg aaaatttgc agatattctct 1801 atagttgttc catatatagg tcttgcttta atataggaa atgagcaca aaaaggaat 1861 Tttaagatg Cacttgaatt Attaggagca ggtattt Tagaatttga Acccgagctt 1921 Tattccta Cattttagt Attcacgata Aatcttttt Taggttcatc Tgataataaa 1981 aaaagtta ttaaagcaat aaataatgca ttgaagaa gagatgaaa atghaagaa 2041 gtatagtt ttatagtatc gattggatg actaaaatta atacacaatt taaaaaga 2101 aaaaaaaaa tgtatcaagc tttacaaaat caagtaaatg cattaaaac attaatagaa 2161 tctagtata atagttatac tttagaggaa aaaatgagc tctaataa atatgattatt 2221 aaagcaatag aaatgaact taatcaaag gttctatag aatgaata tatagacagg 2281 ttcttactg aaagttctat atcctattta atgaattta taatgaagt aaaaatttaat 2341 aattaagag atatgatga gatgtcaa acgtatttat tgaattat tatacacat 2401 ggatcaatct tgggagag tcagcagaa ctaattcta tgtactga taccctaat 2461 atagtattc cttttaagct ttctcttat acagatgata aaattttaat ttcatatttt 2521 aaaattct ttaagaat taaaaattt tcagttttaa attgagata taaaaatgat 2581 aaatacgtag atacttcagg atatgattca atattaata ttaatggaga tgtatattaa 2641 tatccacta aaaaatca atttggaata tataatgata aacttagtga agttaata 2701 tctcaaatg attacattat atatgataat aaatataaaa attttagtat tagttttttgg 2761 gtaagaattc ctaactatga taaatagata gtaatgtta attaatgaata cactatata 2821 attgtaga gagaataata ttcaggatgg aaagtactc ttaatcataa tgaataatt 2881 tggacattgc agataatgc aggattaat caaaattag cattaacta tggtaacgca 2941 aatgtattt ctgattatat aaataagtgg atttttgtaa ctataactaa tgatagatta 3001 ggattcta acctttatat taatggaat ttaatagatc aaaatcaat tttaattta 3061 ggtatattc atgttagtga caatattatta ttaaatag ttaattgtag ttatacaga 3121 tattattggta ttagatattt taatatttt gatataagaat tagatgaac agaattca 3181 actttatata gcaatgacc taatacaat attttgagg atttttgggg aaattatttg 3241 ctttatgaca aagatacta tttaat at gtgttaaaac caataactt tattgatagg 3301 agaaagatt ctactttaag cattaataat atagagca ctattctttt agctaataga 3361 ttatatagtg gaataaagt ttaaaataca agagttaata atagtagtac taacgatat 3421 cttgttagaa agatgatca gttatatatt aatttgtag ccagcaaac tcacttattt 3481 cattatatg ctgatacagc taccacaat aagagaaaa cataaaaat atcatcatct 3541 ggcaatagat ttaatcaagt agtagttatg attcagtag gaaatattg tacaatgaat 3601 tttaaaaata ataatggaaa taatattggg ttgtaggtt tcaaggcaga tactgtagtt 3661 gctagtactt ggtattatac acatatgaga gatcatacaa acagcaatgg atgtttttgg 3721 aactttatt ctgaagaaca tggatggcaa gaaaaaata

[0188] sequence number 2(BoNT / E's polypeptide sequence) PKINSFNYNDPVNDRTILYYIKPGGCQEFYKSFNIMKNIWIIPE RNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGI LLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFET NSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSL HGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLL ADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSF TEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLN PRIITPITGRGLVKKIIRFCKNIVSVKGIRKSICIEINGELFFVASENSYNDDNINT PKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSD IEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKP VQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDA LELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVY SFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDI KQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLNYII QHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMR YKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKN FSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTLQDNAGINQK LAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNIL FKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYL LNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKND QVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMNFKNN NGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

[0189] Sequence number 3 (BoNT / E - UniProt Q00496)

[0190] SEQ ID NO: 4 (Polypeptide sequence of the L chain of BoNT / E) PKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPE RNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGI LLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFET NSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSL HGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGGTDLNIITSAQSNDIYTNLL ADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSF TEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLN PRIITPITGRGLVKKIIRFCKNIVSVKGIR

[0191] SEQ ID NO:5 (Polypeptide sequence of the heavy chain of BoNT / E) KSICIEINNGELFFVASENSYNDDNINT PKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSD IEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKP VQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDA LELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVY SFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDI KQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLNYII QHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMR YKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKN FSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTLQDNAGINQK LAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNIL FKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYL LNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKND QVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMNFKNN NGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK EXAMPLES

[0192] Materials and Methods

[0193] Pharmacodynamic Model

[0194] The pharmacodynamic model utilized the extensor digitorum brevis (EDB) (Figure 1A). The EDB is a superficial muscle of the foot that is easily accessible for electrophysiological studies. It is involved in the extension of the second through fourth toes, and its weakness does not impair gait.

[0195] The EDB muscle has been used previously to determine the effects of BoNT / A as published in Hamjian and Walker (1994), Muscle Nerve, 17(12): 1385-92. Since then, this model has been used extensively to: Quantification of the onset and extent of muscle relaxation in humans following BoNT injection and the duration of effect (Sloop et al (1996), Neurology, 46(5), 1382-6). Comparison of the various subtypes of BoNT (e.g., BoNT / B vs. BoNT / A) (Sloop et al (1997), Neurology, 49(1), 189-94). Confirmation of efficacy of BoNT / A preparations (Park and Ahn (2013), J Clin Neurol, 9(3), 157-164).

[0196] This method involves stimulating the injected EDB and recording its action potentials (Figure 1B). BoNT / E or BoNT / A was administered by ultrasound-guided injection, and surface electrodes were used to stimulate and record action potentials.

[0197] Figure 2 summarizes the dose escalation procedure based on review of safety (blinded) and pharmacodynamic (anonymized) data from previous cohorts.

[0198] Study design and treatment

[0199] A phase 1 randomized, double-blind, placebo-controlled study (human clinical trial) was conducted.

[0200] Each subject received a single intramuscular injection of rBoNT-E or placebo (randomized 3:1) into the right EDB muscle.

[0201] Cohorts of subjects were used, with each cohort injected with a higher dose of rBoNT-E than before, and all subjects in each cohort received the same dose of rBoNT-E or a placebo.

[0202] The maximum number of cohorts was eight. Initial cohort: 4 subjects per cohort (3 rBoNT-E, 1 placebo). Late cohort: When at least three rBoNT-E-injected subjects achieved approximately 50% inhibition of CMAP from baseline at three consecutive time points, the cohort was doubled to eight subjects.

[0203] Cohorts were injected sequentially in ascending dose order. In two consecutive cohorts, dose was escalated until maximal CMAP inhibition (≥85% reduction from baseline) was achieved at three consecutive time points in at least four of six rBoNT-E-injected subjects per cohort. The doses used were 0.1 cell-based assay unit (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).

[0204] Inclusion criteria included the following: Healthy adult males (aged 18-55 years) with a body mass index of 18-30 kg / m 2 . · EDB CMAP total amplitude ≥ 5mV with stimulation of the peroneal nerve for electrophysiological testing (at screening and pre-injection). ·Agree to comply with study protocol and remain in the clinic for the required period of time.

[0205] Exclusion criteria included the following: Previous BoNT treatment within 6 months prior to enrollment. History of hypersensitivity to any component of the rBoNT-E formulation. Any medical condition that would put the subject at risk from exposure to BoNT. Use of neuromuscular transmitters.

[0206] Cell-Based Assays

[0207] The biological activity of rBoNT / E is measured by a cell-based assay (CBA) and expressed as a relative potency percentage against a defined reference standard. This CBA mimics the in vivo mechanism of action of BoNT / E, i.e., internalization of the toxin following binding to the receptor and cleavage of synaptosomal associated protein 25 (SNAP25) by the light chain endopeptidase domain. The CBA uses a mouse neuroblastoma cell line, Neuro-2A, engineered to express a reporter that utilizes full-length SNAP-25 flanked by the fluorophores cyan fluorescent protein and yellow fluorescent protein (YFP). Incubation of engineered cells with BoNT / E results in the toxin binding and internalization via receptor-mediated endocytosis, leading to the release of the BoNT / E light chain into the cytosol. The endopeptidase light chain cleaves SNAP-25 within the reporter, resulting in the release of a C-terminal reporter fragment containing residues of SNAP-25 and YFP into the cytosol. The fragments are rapidly degraded and YFP fluorescence is lost. Recombinant BoNT / E biological activity is only detected when the toxin enters cells via BoNT / E heavy chain receptor binding, internalization, and translocation activity, therefore, the in vitro CBA mimics the natural cell biology of BoNT / E. In the examples below, we list the CBA units that correspond to amounts of BoNT / E.

[0208] One CBA unit can be defined as the amount of BoNT / E required to cleave 1 nM of substrate (SNAP-25) in 1 minute.

[0209] Example 1

[0210] Administration of BoNT / E

[0211] Figure 3 shows that placebo varies around 100% CMAP baseline, demonstrating a clear dose effect of BoNT / E. The two higher doses showed the same pharmacodynamic (PD) profile reaching maximum inhibition (>90%) with a duration of action of approximately 50 days (15% inhibition cutoff) or 30 days (50% inhibition cutoff).

[0212] Administration of Dysport®

[0213] Figure 4 shows that placebo varied around 100% baseline, while Dysport® (BoNT / A) doses of 40 and 70 U reached 60-70% inhibition. Inhibition plateaued over several weeks and did not recover (return to 15% inhibition) at the end of the 26-week follow-up period.

[0214] Example 2

[0215] Analysis up to 26 weeks after administration

[0216] Comparing Figures 4 and 5, it is clear that within the dose range investigated, rBoNT-E has a rapid onset of effect, a large peak effect, and a short duration of effect.

[0217] Example 3

[0218] Onset of action

[0219] The time to onset of action of BoNT / E (defined as the first time point at which 15% CMAP inhibition was recorded) was rapid and consistent across all subjects and all rBoNT-E dose levels studied, and onset was found to range from day 1 (the day of BoNT / E administration) to day 2, whereas for Dysport® it ranged from day 1 to day 7. The results are shown in Figures 6 and 7, and the values ​​are summarized in the table below.

[0220] Table 1 shows the data for rBoNT / E. [Table 1]

[0221] rBoNT / E was associated with a rapid onset of action from day 1 (the day of administration) to day 2. The time to maximum effect was noted from day 2 to day 14 (at the latest). rBoNT / E was also associated with a short duration of action lasting approximately 50 days at the two highest doses tested.

[0222] Table 2 shows the data for Dysport®. [Table 2]

[0223] Dysport® demonstrated a delayed onset of action, occurring between days 1 and 7, across the dose range studied, and a duration of action exceeding 26 weeks after administration.

[0224] Example 4

[0225] Comparison of 40 pg rBoNT / E and 20 U Dysport®

[0226] Figures 8 and 9 show that 40 pg of rBoNT-E produces the same amplitude of effect as 20 U of Dysport®. Specifically, 20 U of Dysport = 108 pg neurotoxin (5.38 pg neurotoxin / U).

[0227] A comparison of PD characteristics is shown in the table below.

[0228] Table 3 shows comparative data for rBoNT / E and Dysport®. [Table 3]

[0229] Example 5

[0230] Comparison of 200 pg rBoNT / E and 40 U Dysport®

[0231] Figures 10 and 11 show that 200 pg of rBoNT-E produces the same amplitude of effect as 40 U of Dysport®. A comparison of the PD characteristics is made in the table below.

[0232] Table 4 shows comparative data for rBoNT / E and Dysport®. [Table 4]

[0233] In conclusion, BoNT / E has been shown to: · Favorable overall safety profile for single intramuscular doses of up to 3.6 ng of rBoNT-E in healthy volunteers (normal muscle). Absence of localized spread to adjacent muscles (AH and ADQ). Rapid onset of action independent of the dose studied. Dose-dependent effect amplitude reaching maximum effect from 0.9ng. - Maximum effect is reached within about one week. Similar PD profiles at the two highest tested doses with duration of effect of approximately 50 days.

[0234] In contrast, inhibition of CMAP was still observed at the end of the 26 week (6 month) recording period with Dysport®.

[0235] Regarding the amplitude of the maximum effect: 20U of Dysport compared to 0.04ng of rBoNT-E 40-70U of Dysport is compared to 0.2ng of rBoNT-E.

[0236] Example 6

[0237] Treatment of upper facial lines

[0238] Subjects with severe glabellar lines (Grade 3 on the Facial Wrinkle Scale (FWS) (0=none, 3=severe)) are presented. rBoNT / E at a dose of at least 0.1 ng (per injection site) is injected intramuscularly at five sites (sequentially so that each muscle is injected substantially simultaneously): one site in the procerus muscle, two sites in each corrugator supercilii muscle.

[0239] The severity of the subject's glabellar lines is assessed on day 7 (post-dosing) using the FWS and is determined to be Grade 0 (thus demonstrating that rBoNT / E has a rapid onset of action in treating upper facial lines).

[0240] The severity of the subject's glabellar lines will be assessed using the FWS on day 35 (post-dose) and determined to be grade 2 (moderate).

[0241] The severity of the subject's glabellar lines is assessed on day 56 (post-dosing) using the FWS and is determined to be grade 3 (severe) (thus demonstrating that rBoNT / E is short-lived in treating upper facial lines).

[0242] All documents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art of biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. [Explanation of symbols]

[0243] [Figure 1] Extensor digitorum brevis: Extensor digitorum brevis stimulus artifact: stimulus artifact Peak-to-peak amplitude: Peak-to-peak amplitude [Figure 2] Percent inhibition of CMAP amplitude: Percent inhibition of CMAP amplitude Maximal inhibition effect reached (85%) in 4 / 8 subjects within 2 consecutive cohorts Stop recruitment / dose escalation: Stop recruitment / dose escalation 8 subjects / cohort (6 active, 2 placebo): Administration staggered by 1 day btw each subject (4 first subjects during a given week), then remaining 4 subjects dosed the week after (2 per day): When 50% inhibition in 2 / 4 subjects: Duplicate the cohort / dose level: 4 subjects / cohort (3 active, 1 placebo): Administration staggered by 1 day btw each subject: Administration is staggered by 1 day between subjects. Dose: Dose Sub-Pharmacological Starting dose 0.1 CBA unit (0.04 ng): Sub-Pharmacological Starting dose 0.1 CBA unit (0.04 ng) Maximal Permitted Dose 9 CBA units (3.6 ng): Maximal Permitted Dose 9 CBA units (3.6 ng) [Figures 3, 5, 6] rBoNT-E time course of effect (mean±sem): Time course of effect of rBoNT / E (mean±sem) EXTENSOR DIGITORUM BREVIS: Extensor digitorum brevis % of baseline CMAP: Percentage of baseline CMAP 15% inhibition: 15% inhibition 50% inhibition: 50% inhibition 85% inhibition: 85% inhibition Placebo: Placebo CBA unit: CBA unit DAY: Day [Figure 4, 7] Dysport time course of effect (mean±sem): EXTENSOR DIGITORUM BREVIS: Extensor digitorum brevis % of baseline CMAP: Percentage of baseline CMAP 15% inhibition: 15% inhibition 50% inhibition: 50% inhibition 85% inhibition: 85% inhibition Placebo: Placebo DAY: Day [Figures 8, 9, 10, 11] Comparative Effects on CMAP Inhibition (mean±sem): EXTENSOR DIGITORUM BREVIS: Extensor digitorum brevis % of baseline CMAP: Percentage of baseline CMAP 15% inhibition: 15% inhibition 50% inhibition: 50% inhibition 85% inhibition: 85% inhibition Toxin E: Toxin E DAY: Day [Figures 12, 13, 14] rBoNT-E time course effect (mean + / - sem): Time course effect of rBoNT / E (mean ± SEM) EXTENSOR DIGITORUM BREVIS: Extensor digitorum brevis CBA unit: CBA unit % of baseline CMAP: Percentage of baseline CMAP DAY: Day

Claims

1. A medicament comprising botulinum neurotoxin serotype E (BoNT / E) for use in the treatment of muscle pain, pain due to muscle spasms, dystonia, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, focal spasticity, or limb spasticity, wherein said medicament, when administered intramuscularly to a human subject, results in maximal inhibition of neurotransmitter secretion from target cells or tissues within 13 days of administration; At 14 days after administration, the inhibition of neurotransmitter secretion from the target cell or tissue is reduced to greater than 25%; the BoNT / E is a recombinant BoNT / E that does not contain complex proteins; the BoNT / E comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3; The BoNT / E is capable of cleaving the 25 kDa synaptosomal associated protein (SNAP-25); The total dose of BoNT / E administered is between 0.04 and 4.0 ng; and The percentage inhibition of neurotransmitter secretion from target cells or tissues decreases by 1-4% per day for each day following maximal inhibition of neurotransmitter secretion.

2. The BoNT / E, when administered, inhibits at least 40% of neurotransmitter secretion from target cells or tissues within 13 days after administration; The pharmaceutical composition of claim 1, which results in a reduction in inhibition of neurotransmitter secretion from the target cell or tissue to 15% or less more than 21 days but less than 100 days after administration.

3. The BoNT / E, when administered, inhibits at least 40% of neurotransmitter secretion from target cells or tissues within 13 days after administration; The pharmaceutical agent according to claim 1 or 2, which results in a reduction in inhibition of neurotransmitter secretion from the target cell or tissue to 15% or less more than 40 days but less than 80 days after administration.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the BoNT / E, when administered, results in a substantially linear decrease in the percentage of inhibition of neurotransmitter secretion after maximum inhibition of neurotransmitter secretion.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the BoNT / E, when administered, results in at least 40% inhibition of neurotransmitter secretion from the target cell or tissue within 10 days of administration.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the BoNT / E, when administered, results in 15% or less inhibition of neurotransmitter secretion from the target cell or tissue within 80 days of administration.

7. The pharmaceutical of any one of claims 1 to 6, wherein the BoNT / E, when administered, results in 15% or less inhibition of neurotransmitter secretion from the target cell or tissue within 60 days of administration.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the BoNT / E, when administered, results in 15% or less inhibition of neurotransmitter secretion from the target cell or tissue within 30 days of administration.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the BoNT / E, when administered, causes greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 100 days.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the BoNT / E, when administered, causes greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 60 days.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the BoNT / E, when administered, causes greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 40 days.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the BoNT / E, when administered, causes greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 30 days.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the BoNT / E, when administered, results in 40 to 60% inhibition of neurotransmitter secretion from target cells or tissues within 13 days of administration.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the BoNT / E, when administered, results in 40 to 60% inhibition of neurotransmitter secretion from target cells or tissues within 11 days of administration.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the percentage of inhibition of neurotransmitter secretion from target cells or tissues is measured using electrophysiological means.

16. The BoNT / E is A pharmaceutical agent according to any one of claims 1 to 15, encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

1.

17. 17. The pharmaceutical of any of claims 1 to 16, wherein the BoNT / E comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:2, with the proviso that the polypeptide sequence comprises one or more of the following amino acids: 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, Here, the numbering of the amino acid positions begins with the N-terminal methionine amino acid residue and ends with the C-terminal amino acid residue of the BoNT / E protein.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the limb spasticity is upper limb spasticity.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the limb spasticity is upper limb spasticity in a subject aged 18 or older.

20. 1. A pharmaceutical composition for use in a cosmetic method for treating upper facial lines comprising intramuscularly administering to a human subject botulinum neurotoxin serotype E (BoNT / E), wherein administering said BoNT / E results in maximal inhibition of neurotransmitter secretion from target cells or tissues within 13 days following administration; At 14 days after administration, the inhibition of neurotransmitter secretion from the target cell or tissue is reduced to greater than 25%; the BoNT / E is a recombinant BoNT / E that does not contain complex proteins; the BoNT / E comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:2 or SEQ ID NO:3; The BoNT / E is capable of cleaving the 25 kDa synaptosomal associated protein (SNAP-25); The total dose of BoNT / E administered is between 0.04 and 4.0 ng; and The percentage inhibition of neurotransmitter secretion from target cells or tissues decreases by 1-4% per day for each day following maximal inhibition of neurotransmitter secretion.

21. The BoNT / E, when administered, inhibits at least 40% of neurotransmitter secretion from target cells or tissues within 13 days after administration; 21. The pharmaceutical composition of claim 20, which results in a reduction in inhibition of neurotransmitter secretion from the target cell or tissue to 15% or less more than 21 days but less than 100 days after administration.

22. The BoNT / E, when administered, inhibits at least 40% of neurotransmitter secretion from target cells or tissues within 13 days after administration; 22. The pharmaceutical composition of claim 20 or 21, which results in a reduction in inhibition of neurotransmitter secretion from the target cell or tissue to 15% or less more than 40 days but less than 80 days after administration.

23. 23. The pharmaceutical composition of any of claims 20 to 22, wherein the BoNT / E, when administered, results in a substantially linear decrease in the percentage inhibition of neurotransmitter secretion after maximal inhibition of neurotransmitter secretion.

24. 24. The pharmaceutical composition of any of claims 20 to 23, wherein the BoNT / E, when administered, results in at least 40% inhibition of neurotransmitter secretion from the target cell or tissue within 10 days of administration.

25. 25. The pharmaceutical composition of any of claims 20 to 24, wherein the BoNT / E, when administered, results in less than 15% inhibition of neurotransmitter secretion from the target cell or tissue in less than 80 days from administration.

26. 26. The pharmaceutical composition of any of claims 20 to 25, wherein the BoNT / E, when administered, results in less than 15% inhibition of neurotransmitter secretion from the target cell or tissue within 60 days of administration.

27. 27. The pharmaceutical composition of any of claims 20 to 26, wherein the BoNT / E, when administered, results in less than 15% inhibition of neurotransmitter secretion from the target cell or tissue within 30 days of administration.

28. 28. The pharmaceutical composition of any of claims 20 to 27, wherein the BoNT / E, upon administration, results in greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 100 days.

29. 29. The pharmaceutical composition of any of claims 20-28, wherein the BoNT / E, when administered, causes greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 60 days.

30. 30. The pharmaceutical composition of any of claims 20-29, wherein the BoNT / E, upon administration, results in greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 40 days.

31. 31. The pharmaceutical composition of any of claims 20 to 30, wherein the BoNT / E, when administered, causes greater than 15% inhibition of neurotransmitter secretion for a period of 5 to 30 days.

32. The pharmaceutical composition of any of claims 20 to 31, wherein the BoNT / E, when administered, results in 40 to 60% inhibition of neurotransmitter secretion from target cells or tissues within 13 days of administration.

33. 33. The pharmaceutical composition of any of claims 20 to 32, wherein the BoNT / E, when administered, results in 40 to 60% inhibition of neurotransmitter secretion from target cells or tissues within 11 days of administration.

34. 34. The pharmaceutical composition of any one of claims 20 to 33, wherein the percentage of inhibition of neurotransmitter secretion from target cells or tissues is measured using electrophysiological means.

35. The BoNT / E is A pharmaceutical composition according to any of claims 20 to 34, encoded by a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

1.

36. 36. The pharmaceutical composition of any of claims 20-35, wherein the BoNT / E comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO:2, wherein the polypeptide sequence comprises one or more of the following amino acids: 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, Here, the numbering of the amino acid positions begins with the N-terminal methionine amino acid residue and ends with the C-terminal amino acid residue of the BoNT / E protein.

37. The pharmaceutical composition according to any one of claims 20 to 36, wherein the upper facial lines are one or more selected from glabellar lines, epicanthal lines, frontal lines, or combinations thereof.

38. 38. The pharmaceutical composition according to any one of claims 20 to 37, for treating glabellar lines.

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