Uses of magnetic nerve stimulation associated with botulinum neurotoxin administration

Abstract

The present invention relates to the use of magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for increasing the therapeutic effect / potency of BoNT, in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT alone. The invention further pertains to the use of magnetic stimulation in combination with BoNT administration for extending the duration of action of BoNT, in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT administration alone. Moreover, the invention is directed to the use of magnetic stimulation in combination with BoNT administration for lowering the incidence of adverse effects of BoNT in the treatment of a disease or disorder in a subject, compared to the treatment of said disease or disorder in said subject by BoNT administration alone.

Description

Uses of magnetic nerve stimulation associated with Botulinum neurotoxin administration

[0001] The present invention relates to the use of magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for increasing the therapeutic effect / potency of BoNT, in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT alone. The invention further pertains to the use of magnetic stimulation in combination with BoNT administration for extending the duration of action of BoNT, in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT administration alone. Moreover, the invention is directed to the use of magnetic stimulation in combination with BoNT administration for lowering the incidence of adverse effects of BoNT in the treatment of a disease or disorder in a subject, compared to the treatment of said disease or disorder in said subject by BoNT administration alone.

[0002] A wide range of adjunct therapies after Botulinum neurotoxin (BoNT) administration has been proposed, in the literature. For instance, the usefulness of physical modalities as adjuvant treatments for managing spasticity has been widely described in the prior art and includes, for example, the anti-spastic effects of extracorporal shock wave therapy (ESWT), therapeutic ultrasound (US), and vibration therapy (VT) (reviewed by Picelli, A., Santamato, A., Chemello, E., Cinone, N., Cisari, C., Gandolfi, M., Ranieri, M., Smania, N., & Baricich, A. (2019). Adjuvant treatments associated with Botulinum neurotoxin injection for managing spasticity: An overview of the literature. Ann Phys Rehabil Med, 62(4), 291-296. https: / / doi.Org / 10.1016 / j.rehab.2018.08.004). WO 2008 / 131941 describes the treatment of movement disorders by a combined use of Botulinum toxin and muscle stimulation, such as electric stimulation, vibration, activation by sound-waves, activation by hydrostatic means, activation by electro-magnetic waves or magnetic fields, or pharmaceutical activation. The study of Shehata et al. (Neurol Sci 36: 1651-1657 (2015)) investigates low-frequency repetitive transcranial magnetic stimulation (rTMS) as a therapeutic tool in corticobasal syndrome (CBS) in combination with Botulinum toxin injection. Yet, these methods are not very effective.

[0003] In pediatrics, BoNT / A is injected in patients suffering from infantile cerebral palsy, for managing spasticity. However, the administration of high doses of BoNT / A can result in systemic side effects, in such patients (Multani I, Manji J, Hastings-Ison T, Khot A, Graham K. Botulinum Toxin in the Management of Children with Cerebral Palsy. Paediatr Drugs. 2019 Aug;21(4):261-281. doi: 10.1007 / s40272-019-00344-8. PMID: 31257556; PMCID: PMC6682585, and references cited therein). Also, the treatment of patients with cervical dystonia using high doses of Botulinum neurotoxin can cause severe side effects (Petracca M, Lo Monaco MR, lalongo T, Di Stasio E, Cerbarano ML, Maggi L, De Biase A, Di Lazzaro G, Calabresi P, Bentivoglio AR. Efficacy and safety of long-term botulinum toxin treatment for acquired cervical dystonia: a 25-year follow-up. J Neurol. 2023 Jan;270(l):340-347. doi: 10.1007 / s00415-022-11343-0. Epub 2022 Sep 6. PMID: 36068376; PMCID: PMC9813057). In such or other cases requiring the application of high doses of Botulinum neurotoxin, it can be desirable to reduce the dose of Botulinum neurotoxin (BoNT) or to enhance the effects of Botulinum neurotoxin without dose increase, in order to reduce or even prevent undesired side effects in Botulinum neurotoxin treatments.

[0004] In view of the above, there is still a strong need for more appropriate and effective adjuvant treatment protocols for Botulinum neurotoxin administration, especially for those diseases or disorders that require high doses of Botulinum neurotoxin causing adverse effects.

[0005] Thus, the technical problem underlying the present invention may be seen as the provision of means and methods complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0006] The present invention relates to the use of magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for increasing the therapeutic effect / potency of Botulinum neurotoxin (BoNT), in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT alone. Further, the invention pertains to the use of magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for extending the duration of action of BoNT, in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT administration alone. In addition, the invention is directed to the use of magnetic stimulation in combination withBotulinum neurotoxin (BoNT) administration for lowering the incidence of adverse effects of Botulinum neurotoxin (BoNT) in the treatment of a disease or disorder in a subject, compared to the treatment of said disease or disorder in said subject by BoNT administration alone, preferably in cases which require the application of high doses of Botulinum neurotoxin.

[0007] This is the first report on an adjuvant treatment in which repetitive magnetic stimulation is used in association with Botulinum neurotoxin (BoNT) administration for increasing the potency of BoNT for better managing a disease or disorder in a subject, to the inventors’ best knowledge. Magnetic stimulation is a non-invasive method using magnetic energy to modulate the function of the nervous system. The stimulation can apply over the brain, spinal cord, nerve roots or peripheral nerves and muscles. Magnetic stimulation of peripheral nerves results in an action potential in these nerves and corresponding peripheral muscle activity. This muscle activity is the result of an increased release of acetylcholine from nerve terminals at the neuromuscular junctions. If Botulinum neurotoxin (BoNT) is injected into this area, it enters the neurons by endocytosis, blocks acetylcholine release which in turn results in muscle paralysis. Surprisingly, it has been found by the present inventors that BoNT / A treatment in combination with repetitive peripheral (or transcutaneous) magnetic stimulation resulted in increased effectiveness / potency of BoNT / A, compared to BoNT / A treatment without magnetic stimulation, in a mouse model, as demonstrated in the following Examples. Clinically, this means that lower doses of Botulinum neurotoxin (BoNT) can be used in order to achieve the same therapeutic effect or that an enhanced effect can be accomplished with the same Botulinum neurotoxin (BoNT) dose, using magnetic stimulation after Botulinum neurotoxin (BoNT) administration, preferably directly after BoNT administration as defined herein. The low-dose combination therapy usage has greater efficacy and less toxicity than using a higher dose of BoNT. Consequently, the use of lower doses of BoNT in combination therapy with magnetic stimulation may be associated with a lower incidence of adverse effects of individual drugs. Accordingly, the present invention can be advantageously used for reduction of the Botulinum neurotoxin (BoNT) dose, or for increasing the Botulinum neurotoxin (BoNT) therapeutic effect without raising the dose. As appreciated by those skilled in the art, the uses of the present invention are particularly appropriate for treatment of diseases or disorders which need the administration of high doses of Botulinum neurotoxin (BoNT) causing severe side effects in the patients, such as the treatment of infantile cerebral palsy, cervical dystonia, spasticity, especially spasticity of large muscle areas or groups, or the treatment of patients having an overactive bladder.They can further be utilized for stroke rehabilitation, such as for improvement of the motor function or for motor function recovery of extremities of stroke patients, reduction of muscle hypertension or stiffness and improvement of muscular flexibility, or for improvement of muscle function of patients with central paralysis, of chronic pain, migraine, or spinal cord injury (regeneration), as set forth elsewhere herein. In light of this, magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration can also be used for reduction or prevention of undesired side effects in Botulinum neurotoxin treatments.

[0008] In one embodiment of the uses of the present invention, magnetic stimulation is repetitive magnetic stimulation.

[0009] Magnetic stimulation can be used to elicit excitatory and inhibitory action potentials, both in the peripheral and central nervous system. Due to the fact that magnetic stimulation, in contrast to electrical stimulation, allows for evoking action potentials without inducing pain and this even via an intact scull, this method is widely applied in clinical situations and for research purposes. The existing areas in which magnetic stimulation is applied both in human and veterinary medicine does by far not exhaust the vast possibilities for exploiting this method of stimulation (Dissertation by Daniela Emrich, 2009: Transkranielle, spinale und periphere Magnetstimulation bei Maus und Hund. Ludwig-Maximilians-Universitat Munchen, Germany). Repetitive magnetic stimulation is performed utilizing a magnetic stimulator that is capable of delivering not only single magnetic pulses but also duplicate or triplicate pulses or even series of pulses such as bursts, to a subject (Huang et al., Neuron. 2005 Jan 20;45(2):201-6; Dissertation by Daniela Emrich, 2009, loc. cit.).

[0010] In still another embodiment of the uses of the present invention, the magnetic stimulation is repetitive transcutaneous magnetic stimulation or repetitive transcranial magnetic stimulation. Repetitive transcutaneous magnetic stimulation is preferably over muscles and / or nerves such as peripheral nerves. As acknowledged by the skilled person, peripheral nerves encompass spinal nerves. Repetitive transcutaneous magnetic stimulation or repetitive peripheral magnetic stimulation - both expressions are used interchangeably herein - has been utilized e.g. for reduction of muscle hypertension or muscle stiffness and improvement of muscular flexibility (Krewer, C., Hartl, S., Muller, F., & Koenig, E. (2014). Effects of repetitive peripheral magnetic stimulation on upper-limb spasticity and impairment in patients with spastic hemiparesis: a randomized, double-blind, sham-controlled study. Arch Phys Med Rehabil, 95(6), 1039-1047. https: / / doi.Org / 10.1016 / j.apmr.2014.02.003; Werner, C. S., M.; Wernicke, S.; Bryl, B., Hesse, S. (2016). Repetitive Peripheral Magnetic Stimulation (rpMS) in Combination with Muscle Stretch Decreased the Wrist and Finger Flexor Muscle Spasticity in Chronic Patients after CNS Lesion. Int J Phys Med Rehabil, 6. https: / / doi.org / 10.4172 / 2329- 9096.1000352; Zschorlich, V. R., Hillebrecht, M., Tanjour, T., Qi, F., Behrendt, F., Kirschstein, T., & Kohling, R. (2019). Repetitive Peripheral Magnetic Nerve Stimulation (rPMS) as Adjuvant Therapy Reduces Skeletal Muscle Reflex Activity. Front Neurol, 10, 930. https: / / doi.org / 10.3389 / fneur.2019.00930; Nielsen et al., Mult Scler. 1996 Dec;2(5):227-32), or for improvement of muscle function of patients with central paralysis (Heldmann, B., Kerkhoff, G., Struppler, A., Havel, P., & Jahn, T. (2000). Repetitive peripheral magnetic stimulation alleviates tactile extinction. Neuroreport, 11(14), 3193- 3198. https: / / doi.org / 10.1097 / 00001756-200009280-00029). The study by Chalfouh et al. analyzed the regenerative effect of trans-spinal magnetic stimulation after spinal cord injury (Neurotherapeutics. 2020 Oct; 17(4): 2069-2088).

[0011] Repetitive transcranial magnetic stimulation (rTMS) is a treatment technique that uses a magnetic field to influence brain activity, so it focuses on the magnetic stimulation of the brain. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique approved by U.S. and European guidelines for the management of treatmentresistant depression (TRD). Current evidence has established rTMS as an effective and safe technique, and thus an alternative to conventional methods (Rossi et al., Clin Neurophysiol. 2009 Dec;120(12):2008-2039; Baeken et al., Curr Opin Psychiatry. 2019 Sep; 32(5): 409-415; Sonmez et al., Psychiatry Res. 2019 Mar; 273: 770-781). Repetitive transcranial magnetic stimulation is based on the Faraday's electromagnetic induction principle. The electric current passes through a magnetic coil and initiates a magnetic field. This field, in interaction with high electrically conductive tissues, such as the brain, induces a secondary electric field. The magnetic field passes through the skin, bone, and adipose tissue (tissues with low electrical conductivity), essentially without resistance and with little deflection, and consequently, the induced current is focal. Many studies have used repetitive transcranial magnetic stimulation (rTMS) to induce long-lasting changes in the excitability of cortical and corticospinal pathways after incomplete spinal cord injury (iSCI) (de Araujo AVL, Barbosa VRN, Galdino GS, Fregni F, Massetti T, Fontes SL, de Oliveira Silva D, da Silva TD, Monteiro CBM, Tonks J, Magalhaes FH. Effects of high- frequency transcranial magnetic stimulation on functional performance in individuals with incomplete spinal cord injury: study protocol for a randomized controlled trial. Trials. 2017Nov 6;18(1):522. doi: 10.1186 / sl3063-017-2280-l . PMID: 29110687; PMCID: PMC5674824.). Repetitive transcranial magnetic stimulation has also been used in stroke rehabilitation (Xu AH, Sun YX. Research hotspots and effectiveness of repetitive transcranial magnetic stimulation in stroke rehabilitation. Neural Regen Res. 2020 Nov;15(l l):2089-2097. doi: 10.4103 / 1673-5374.282269. PMID: 32394967).

[0012] However, so far none of the studies relating to transcutaneous (peripheral) or transcranial magnetic stimulation has shown that BoNT treatment in combination with repetitive peripheral (or transcutaneous) magnetic stimulation resulted in increased effectiveness / potency of said BoNT, compared to BoNT treatment without magnetic stimulation. Magnetic stimulation in combination with BoNT / A administration for therapeutic uses is illustrated by the following Examples, in a mouse model.

[0013] In a further preferred embodiment of the uses of the present invention, magnetic stimulation is performed using one or more magnetic coil(s) connected to a magnetic stimulator.

[0014] Magnetic stimulators and magnetic coils for magnetic stimulation are well described in the literature (see e.g. the references to magnetic stimulation cited herein) and are also commercially available, such as the Magnetic Stimulator MagPro Compact or Magnetic Stimulator Magstim 2002provided by Micromed. Further providers for magnetic stimulators and coils include MagVenture (with e.g. magnetic stimulator Mag Pro R30 or Mag Pro XI 00), MAG & More, Magstim, Neurolite AG, and others. Frequently used coils for magnetic stimulation include, for instance circular coils, figure-of-eight (FoE) coils, butterfly (V-shape) coils, cone (double circular) coils, downward-bending U-shape (DBU) coils, upward-bending U-shape (UBU) coils, halo coils, and hesed coils (H-coils). Commercially available magnetic coils include, for instance, Cool-B65 A / P, Cool-B65 A / P RO, Cool-B70 A / P, C00I-D-B8O A / P or MMC-140 A / P (Magventure). For example, an eight-shaped double coil connected to the Magstim rapid stimulator or an MMC-140 A / P coil connected to a MagPro XI 00 stimulator (Magventure GmbH, Willich, Germany) can be used for magnetic stimulation in a human subject. Magnetic stimulators for animals such as mice or dogs are described, for instance, in the Dissertation by Daniela Emrich, 2009, loc. cit.

[0015] In a still further preferred embodiment of the uses of the present invention, the magnetic stimulation is performed after botulinum neurotoxin (BoNT) administration.Preferably, the magnetic stimulation is performed directly after BoNT administration, such as 1 second to 60 minutes after botulinum neurotoxin (BoNT) administration, more preferably 1, 2, 3, 5, 10, 20 or 30 second(s), 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes or 60 minutes after BoNT administration, even more preferably between 1 second and 10 minutes or between 5 seconds and 5 minutes or between 10 seconds and 2 minutes after BoNT administration.

[0016] In yet another preferred embodiment of the uses of the present invention, the following parameter settings can be used for the magnetic stimulation: A peak to peak magnetic flux density on a coil surface of at least 0.2 T, 0.4 T, 1.5 T, 2 T, or at least 3 T, up to 7 T. The repetition rate may exceed 0.1 Hz, 0.5 Hz, 1 Hz, 10 Hz, 20 Hz, 30 Hz, 40 Hz 50 Hz, 80 Hz, 90 Hz, 100 Hz or 120 Hz, and up to 150 Hz, with preferable repetition rate up to 700 Hz. The initial or successive treatments can last several seconds or longer, for example, for at least 5, 10, 30, 60, 120 or 240 seconds, or longer. The pulse width is in the range of tens to hundreds of microseconds. Preferably, a frequency of 0.1 to 50 Hz, and an intensity of 1 to 130% is used. More preferably, a frequency of 0.1 to 50 Hz, an intensity of 1 to 130%, 1 to 10 pulses, in 1 to 50 serial pulse blocks is used. Protocols and parameter settings for magnetic stimulation such as for frequency, intensity, pulse number, pulse sequence, sequence duration, sequence interval, stimulation time, etc. or using low frequency / high frequency, are well described in the literature, such as in the references cited herein (see e.g. Rossi et al., Clin Neurophysiol. 2009 Dec;120(12):2008-2039; Dissertation by Daniela Emrich, 2009: Transkranielle, spinale und periphere Magnetstimulation bei Maus und Hund. Ludwig-Maximilians-Universitat Munchen, Germany; Chalfouh et al., Neurotherapeutics. 2020 Oct; 17(4): 2069-2088; EP3698847B1) or in manuals by the commercial providers of magnetic stimulators and magnetic coils. For transcranial magnetic stimulation, theta burst stimulation (TBS) is preferably used (Ozdemir et al., Ideggyogy Sz. 2021 Jan 30;74(l-2):41-49. doi: 10. 18071 / isz.74.0041; Lowe et al., Neuropsychologia, Volume 111, March 2018, Pages 344-359). Preferably, a frequency of 1 to 100 Hz, and an intensity of 1 to 140%, is utilized for TBS.

[0017] In a further preferred embodiment of the uses of the present invention, Botulinum neurotoxin administration is via intramuscular administration. Preferably, Botulinum neurotoxin administration is via intramuscular injection.

[0018] In a still further preferred embodiment of the uses of the present invention, Botulinum neurotoxin (BoNT) is administered in a dose of 1-30 U / kg body weight inmouse, preferably 10-30 U / kg, more preferably 20-30 U / kg, and in a dose of 1-24 U / kg body weight in human, preferably 10-16 U / kg.

[0019] In another preferred embodiment of the uses of the present invention, the botulinum neurotoxin is BoNT / A, BoNT / Al, BoNT / A2, B0NT / A6, BoNT / B, BoNT / Cl, BoNT / D, BoNTZE, BoNT / F, or BoNT / G. Preferably, the botulinum neurotoxin is BoNT / A, BoNT / A2, or B0NT / A6, more preferably BoNT / A, and even more preferably XEOMIN® (IncobotulinumtoxinA: NT201; Merz Pharma GmbH & Co. KGaA). Preferably, BoNT / A, more preferably NT201, is administered in a dose of 10-16 U / kg body weight in human.

[0020] A Botulinum neurotoxin (BoNT) as referred to herein encompasses BoNT / A, BoNT / Al, BoNT / A2, B0NT / A6, BoNT / B, BoNT / Cl, BoNT / D, BoNTZE, BoNT / F, BoNT / G, and XEOMIN® (IncobotulinumtoxinA: NT201; Merz Pharma GmbH & Co. KGaA). IncobotulinumtoxinA (Xeomin®; also known as NT 201) is a purified form of botulinum toxin type A (BoNT / A) that contains no accessory proteins and has a high specific biological activity (Frevert J. Xeomin is free from complexing proteins. Toxicon. 2009; 54: 697-701; Frevert J. Content of botulinum neurotoxin in botox® / vistabel®, dysport® / azzalure®, and xeomin® / bocouture® Drugs R D. 2010; 10: 67-73; Frevert J, Dressier D. Complexing proteins in botulinum toxin type A drugs: a help or a hindrance? Biologies. 2010; 4: 325-332; Dressier D. Five-year experience with IncobotulinumtoxinA (Xeomin((®)): the first botulinum toxin drug free of complexing proteins. Eur J Neurol. 2012; 19: 385-389). The Botulinum neurotoxin (BoNT) polypeptide and, in particular, its light chain and heavy chain are derivable from one of the antigenically different serotypes of Botulinum neurotoxins indicated above. In an aspect, said light and heavy chain of the Botulinum neurotoxin polypeptide are the light and heavy chain of a neurotoxin selected from the group consisting of: BoNT / A (1296aa, accession number P0DPI0.1, GI: 1431907642), BoNT / Al (1296aa, UniProt C9WWY7; GenBank accession number AM412317), BoNT / A2 (1296aa, accession number Q45894.3, GI: 20137335), B0NT / A6 (1296aa, GenBank: ACW83608.1), BoNT / B (1291aa, accession number P10844,3, GI: 399134), BoNT / Cl (1291aa, accession number P18640.3, GI: 1431906477), BoNT / D (1276aa, accession number P19321.1, GI: 115188), BoNTZE (125 laa, accession number Q00496.2, GI: 399135), BoNT / F (1274aa, accession number A7GBG3.1, GI: 1431907643), and BoNT / G (1297aa, accession number Q60393.2, GI: 2499920). The amino acid sequences of said Neurotoxin polypeptides and polynucleotides encoding them are known and well described in the art; see e.g. SEQ ID Nos. 1 to 16 of WO 2014 / 207109; Barash & Arnon 2014, JID 209: 183-191; Dover et al. 2014, JID209: 192-202; Maslanka et al., J Infect Dis. 2016 Feb l;213(3):379-85. doi: 10.1093 / infdis / jiv327. Epub 2015 Jun 10; Moritz et al., mSphere. 2018 Sep-Oct; 3(5): e00466-18). The said polynucleotides can also be a variant of the aforementioned polynucleotides comprising one or more nucleotide substitutions, deletions and / or additions which in still another aspect may result in a polypeptide having one or more amino acid substitutions, deletions and / or additions. Moreover, a variant polynucleotide shall in another aspect comprise a nucleic acid sequence variant being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence as shown in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or 15 of WO 2014 / 207109, or a nucleic acid sequence variant which encodes an amino acid sequence being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16 of WO 2014 / 207109. The term "identical” as used herein refers to sequence identity characterized by determining the number of identical amino acids between two nucleic acid sequences or two amino acid sequences wherein the sequences are aligned so that the highest order match is obtained. It can be calculated using published techniques or methods codified in computer programs such as, for example, BLASTP, BLASTN or FASTA (Altschul 1990, J Mol Biol 215, 403). The percent identity values are, in one aspect, calculated over the entire amino acid sequence. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (Higgins 1989, CABIOS 5, 151) or the programs Gap and BestFit (Needleman 1970, J Mol Biol 48; 443; Smith 1981, Adv Appl Math 2, 482), which are part of the GCG software packet (Genetics Computer Group 1991, 575 Science Drive, Madison, Wisconsin, USA 53711), may be used. The sequence identity values recited above in percent (%) are to be determined, in another aspect, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. In an aspect, each of the aforementioned variant polynucleotides encodes a polypeptide retaining one or more and, in another aspect, all of the biological properties of the respective Botulinum neurotoxin polypeptide, i.e. BoNT / A, BoNT / Al, BoNT / A2, B0NT / A6, BoNT / B, BoNT / Cl, BoNT / D, BoNT / E, BoNT / F, and BoNT / G. Those of skill in the art will appreciate that full biological activity is maintained only after proteolytic activation, even though it is conceivable that the unprocessed precursor can exert somebiological functions or be partially active. "Biological properties" or “biological functions” refers to (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and / or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle membrane fusion. In vivo assays for assessing biological activity include the mouse LD50 assay and the ex vivo mouse hemidiaphragm assay as described by Pearce et al. (Pearce 1994, Toxicol. Appl. Pharmacol. 128: 69-77) and Dressier et al. (Dressier 2005, Mov. Disord. 20: 1617-1619, Keller 2006, Neuroscience 139: 629-637). The biological activity of a Botulinum neurotoxin is commonly expressed in Units (U), in human. In mouse, the biological activity is usually indicated in Mouse Units (MU). For instance, 1 MU is the amount of Botulinum neurotoxic component, which kills 50% of a specified mouse population after intraperitoneal injection, i.e. the mouse i.p. LD50.

[0021] Another useful method for determining the biological activity (biological potency) of a botulinum neurotoxin is a cell-based potency assay as it is disclosed, for example, in W02009 / 114748, WO 2013 / 049508 or WO 2014 / 207109. The activity results obtained with such cell-based assays correspond to the activity values obtained in the mouse i.p. LD50 assay because the values are calibrated using the LD50 reference standard. Due to differences in the LD50 tests used by manufacturers of commercial Botulinum neurotoxin formulations, the unit potencies indicated by the manufacturers for their commercial Botulinum neurotoxin formulations is proprietary and cannot easily be compared. Therefore, within the framework of the present invention, the conversion rates provided below are used to establish the comparative potencies of incobotulinumtoxinA ("INCO"; Xeomin®, Bocouture®; botulinum toxin serotype A, free of complexing proteins; Merz Pharmaceuticals GmbH), onabotulinumtoxinA ("ONA"; Botox®, Vistabel®; botulinum toxin complex of serotype A; Allergan Inc.), abobotulinumtoxinA ("ABO"; Dysport®, Azzalure®; botulinum toxin complex of serotype A; Medicis Pharmaceutical Corp., Galderma Lab.) and rimabotulinumtoxinB ("RIM"; Myobloc®, NeuroBloc®; botulinum toxin serotype B; Solstice Neurosciences Inc.). For use herein, the conversion rate of ONA and INCO is 1 : 1. The conversion rate of ONA / INCO:ABO is 1 :2.5-1 :3.5. The conversion rate of ONA / INCO:RIM is 1 :50. Furthermore, within the context of the present invention, 1 U of INCO (Xeomin®) and 1 U of onabotulinumtoxinA ("ONA"; Botox®) shall be deemed to correspond to one mouse LD50 (1.0 LD50) unit, or 1 U, measured as described above.

[0022] In yet another preferred embodiment of the uses of the present invention, the subject is a mammal such as a human or an animal. An animal can be a mouse or a rat, adomestic animal such as s cat, dog, rabbit, fish, hamster or guinea pig, or a farm animal such as donkey, cow, sheep, horses, goat, Arabian camel, Bactrian camel, llama and alpaca, donkey, reindeer, water buffalo, yak, Bali cattle, and Mithan, and a pig. Preferably, the subject is mouse or human, more preferably human.

[0023] In a further preferred embodiment of the uses of the present invention, magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration, including also transcranial magnetic stimulation such as repetitive transcranial magnetic stimulation in combination with Botulinum neurotoxin (BoNT), causes a muscle contraction in the subject, as a readout. Herein, a visible muscle contraction indicates a strong muscle activation. However, an invisible muscle contraction detected by electromyography (EMG) can also be used as a readout, as appreciated by those of skill in the art (Robertson and Galbadon, Integr Comp Biol. 2008 Aug; 48(2): 312-320; Fukuhara et al., Scientific Reports volume 11, Article number: 21208 (2021)). Preferably, magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration causes a visible muscle contraction in the subject, as shown in the following Examples. In comparison, no such visible muscle contraction could be observed in the studies by Emrich (Dissertation by Daniela Emrich, 2009: Transkranielle, spinale und periphere Magnetstimulation bei Maus und Hund. Ludwig-Maximilians-Universitat Munchen, Germany).

[0024] In a still further preferred embodiment of the uses of the present invention, said combination of magnetic stimulation and BoNT administration is for treatment of infantile cerebral palsy, cervical dystonia, spasticity, preferably spasticity of large muscle areas or groups, an overactive bladder, for stroke rehabilitation, such as for improvement of the motor function or for motor function recovery of extremities of stroke patients, reduction of muscle hypertension or stiffness and improvement of muscular flexibility, or for improvement of muscle function of patients with central paralysis, of chronic pain, migraine, or for regeneration of spinal cord injury.

[0025] Cerebral palsy or infantile cerebral palsy (Colver et al., Lancet. 2014 Apr 5;383(9924): 1240-9; Graham et al., Nat Rev Dis Primers. 2016 Jan 7:2: 15082; Faccioli et al., Front Neurol. 2023 May 25; 14:1171224) means a non-progressive motor disorder that occurs at a rate of 2 per 1000 live births. Cerebral palsy is characterized by an absence or delay in motor development, abnormal muscle tone (often spasticity), impaired control of movement, abnormal posture, and in many cases, contractures. Other associated conditions may include gastro-esophageal reflux, epilepsy, visual impairment, respiratory problems,drooling and uncoordinated swallowing - all factors that may contribute to sleep deficiency. Between 23% and 46% of children with cerebral palsy experience sleep problems. Many of the sensori-motor and cognitive features of cerebral palsy (such as immobility, pain, and seizures) act as predisposing factors for sleep problems in this population. Nocturnal polysomnography in children with cerebral palsy may need to be combined with an extended 16-channel electroencephalography montage if there is a suspicion for nocturnal seizures. Management is best accomplished through an interdisciplinary approach - for example, obstructive sleep apnea (OSA) may require adenotonsillectomy, neurogenic dysphagia may necessitate gastrostomy tube placement, and excessive oral secretions may require Botulinum neurotoxin injections to the salivary gland. Treatment of spasticity with intrathecal baclofen has been shown to improve sleep by decreasing apneic events and minimizing involuntary movements.

[0026] Cervical dystonia (CD) (Erro et al., Toxins (Basel). 2023 Jun 9; 15(6):391 ; Boyce et al., Neurol Sci 2022 Aug;43(8):4663-4670) is the most common form of the focal dystonia, which causes abnormal neck posture. CD can present with neck spasm, abnormal neck posture, head and neck tremor, and pain. The exact pathophysiology of CD is unknown. There are several hypotheses explaining the role of basal ganglia in CD. Dystonic movements may be sustained or spasmodic and may remain focal or spread to adjacent muscles. Remissions may rarely occur. Many symptomatic treatments are available, including medical and surgical therapy and as well as local injections of botulinum toxin into involved neck muscles. Currently, Botulinum neurotoxin injection is the most effective symptomatic therapy.

[0027] Spasticity (Tamburin et al., Curr Opin Neurol. 2022 Dec l;35(6):728-740; Zhou et al., Front Neurosci. 2023 Jul 18;17:1177283; Otero-Romero et al., Mult Scler. 2016 Oct;22(l l): 1386-1396) is a disorder of sensorimotor control, resulting from an upper motor neuron (UMN) lesion, presenting as intermittent or sustained involuntary activation of muscles. Spasticity is a result of a variety of damages to the spinal cord or the brain, of which the most common are: ischemic strokes, cerebral hemorrhage, traumatic brain injury (TBI), and spinal cord injury (SCI). Furthermore, spasticity occurs in neuroinflammatory diseases such as multiple sclerosis (MS) or neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Spasticity is one of the most common causes of physical disability worldwide, with an estimated incidence of at least 12 million patients. Spasticity develops in about 35% of patients after stroke, 90% with cerebral palsy, 50% with TBI, 40% with SCI, and 37% - 78% with MS. Moreover, spasticity-related movementimpairments go along with the risk of falling and resulting fractures, associated with increased morbidity and mortality, and particularly impact elderly patients.

[0028] An overactive bladder (Truzzi et al., Neurourol Urodyn. 2022 Mar;41(3):710-723; Hu et al., Toxins (Basel). 2023 Feb 17; 15(2): 166; Scameciu et al., Exp Ther Med. 2021 Dec;22(6): 1444) means a common disabling condition that affects health-related quality of life. The International Continence Society (ICS) derived a consensus symptomatic definition of overactive bladder syndrome as urinary urgency, with or without urge incontinence, usually with urinary frequency and nocturia, in the absence of pathologic and metabolic factors that would explain the symptoms. Urodynamically, overactive bladder is characterized by the presence of involuntary bladder contractions that occur during bladder filling despite the patient's attempt to suppress them. The National Overactive Bladder Evaluation (NOBLE) study was initiated to better understand the prevalence and impact of overactive bladder in a broad spectrum of the U.S. population. Using a clinically validated, computer-assisted telephone interview questionnaire, a sample population of adults, who were 18 years old or older and representative of the main population by sex, age, and geographic region, was surveyed. A surprising result from this study is the equal prevalence of urgency-related bladder control problems in men and women (16.0% and 16.9%, respectively), although more women (13.4%) suffer from overactive bladder with incontinence than men (2.6%).

[0029] Stroke (Diener et al., Nat Rev Neurol. 2022 Aug;18(8):455-465; Arienti et al., PLoS One. 2019 Jul 19;14(7):e0219781) is a generic term for cerebrovascular accident (CVA) resulting in a sudden or rapidly progressing neurological defect, which does not resolve within 24 hours. Stroke is the third highest cause of death in the UK, after ischaemic heart disease and all cancer types combined, with around 150000 people having a stroke per year. The effects of stroke principally result in unilateral numbness, weakness and partial or complete paralysis of the arm, leg or face on the contralateral side of the brain.

[0030] Central paralysis (Heldmann, B., Kerkhoff, G., Struppler, A., Havel, P., & Jahn, T. (2000). Repetitive peripheral magnetic stimulation alleviates tactile extinction. Neuroreport, 11(14), 3193-3198. https: / / doi.org / 10.1097 / 00001756-200009280-00029; Struppler A, Jakob C, Muller-Barna P, Schmid M, Lorenzen HW, Prosiegel M, Paulig M. Eine neue Methode zur Fruhrehabilitation zentral— bedingter Lahmungen von Arm und Hand mittels Magnetstimulation, Zeitschrift fur EEG und EMG 1996; 27: 151-157;Struppler A, Havel PM, Muller-Barna P, Lorenzen, HW. Eine neue Methode zur Rehabilitation zentraler Lahmungen von Arm und Hand mittels peripherer Magnetstimulation. Neurologic und Rehabilitation 1997; 3: 145-158; A. Struppler, B. Angerer, B. Gebhard, Neuro Rehabil 2009; 15 (1): 28-38; Bernhardt M, Angerer B, Buss M, Struppler A: Nonlinear System Identification in Stroke Rehabilitation. Automatisierungstechnik 2007; 55: 11, DOI 10.1524 / auto.2007.55.11.570) means the central or also spastic form of paralysis which is characterized by the involvement of the central motor neuron. The lesion is therefore located in the CNS, either in the brain or in the spinal cord. The etiology can be varied, e.g.: strokes, tumors, inflammation, trauma, subdural or epidural hematomas, etc. Depending on the severity and localization, one can distinguish between plegia (complete paralysis) and paresis (only partial paralysis).

[0031] Botulinum neurotoxins (BoNTs) bind irreversibly to the presynaptic surface of cholinergic nerve terminals in the neuromuscular junctions, which results in the inhibition of acetylcholine release, thereby inducing muscle weakness. The mentioned neurotoxins enter the neuron by endocytosis and then interact with the soluble N-ethylmaleimide- sensitive factor attachment protein receptor (SNARE) apparatus to disrupt acetylcholine release. The reversible blockage of acetylcholine release from nerve terminals at the neuromuscular junction by BoNT / A results in a preliminary muscle paralysis. The paralytic effects of BoNT / A are, however, time-limited and require repeated injections at regular intervals to achieve long-term therapeutic benefits. Botulinum neurotoxin (BoNT) treatment of some indications such as infantile cerebral palsy, spasticity, especially spasticity of large muscle areas or groups, cervical dystonia or overactive bladder have many side effects which vary. For example, administration of high doses of Botulinum neurotoxin (BoNT) for managing spasticity can cause systemic muscle weakness, whereas this administration can induce dysphagia in BoNT treatment of cervical dystonia and can result in the production of neutralizing antibodies in therapeutic management of overactive bladders. Evidently, Botulinum neurotoxin (BoNT) treatment has a narrow therapeutic range, under these conditions. Magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration according to the uses of the present invention advantageously increases the efficacy and duration of action of Botulinum neurotoxin (BoNT), reduces the severity and / or rate of adverse events, improves the quality of life of the patients, and results in better product safety. Moreover, the benefit of magnetic stimulation lies in broad availability of appropriate magnetic stimulators and magnetic coils in neurology. In addition, magnetic stimulation is a non-invasive and painless method.

[0032] Typically, the Botulinum neurotoxin as used within the present invention is in the form of a liquid composition. The liquid composition can be formulated by various techniques dependent on the desired application, as known in the art. It may be provided as a ready-to-use liquid formulation or in the form of a lyophilized powder that is to be reconstituted, typically in physiological saline, prior to use. Preferably, the Botulinum neurotoxin used within the present invention is in the form of an aqueous solution, more preferably a saline solution or a physiological saline solution, and most preferably a phosphate buffered physiological saline solution. The aqueous solution may additionally comprise one or more pharmaceutically acceptable substances. Suitable pharmaceutically acceptable substances comprise those well known in the art, see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0033] In particular, the aqueous Botulinum neurotoxin solution or composition may include other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. Thus, the aqueous Botulinum neurotoxin composition may contain glycerol, protein stabilizers (e.g., HSA) or non-protein stabilizers such as polyvinyl pyrrolidone (PVP), hyaluronic acid or free amino acids, e.g., methionine or histidine. Suitable nonproteinaceous stabilizers are disclosed in WO 2005 / 007185 or WO 2006 / 020208. Also, it may be free of amino acids and / or free of stabilizing peptides (e.g., consisting of 5 to 50 amino acids, 10 to 40 amino acids or 15 to 30 amino acids). The Botulinum neurotoxin composition can also include a non-ionic or ionic surfactant, e.g., polysorbate or poloxamer. A suitable formulation for HSA-stabilized formulation comprising a Botulinum neurotoxin used within the present invention is, for example, disclosed in US 8,398,998 B2.

[0034] Preferably, the Botulinum neurotoxin used within the present invention is in the form of an aqueous solution comprising sodium chloride (NaCl), preferably in the form of a physiological saline solution (i.e. a solution including sodium chloride in physiological concentration, e.g., about 9 g / 1 NaCl), which further comprises one or more of the following (i) to (ix): (i) no other excipient (except NaCl), (ii) human serum albumin (HSA) and a sugar, in particular a monosaccharide or a disaccharide, (iii) human serum albumin (HSA) and lactose, (iv) human serum albumin (HSA) and sucrose, (v) a monosaccharide and / or a disaccharide (e.g. lactose and / or sucrose), (vi) no buffer, (vii) no single amino acids, (viii) no human serum albumin (HSA), sodium chloride and lactose or no HSA,sodium chloride and sucrose, or (ix) no HSA and sodium chloride, or any combination of (i) to (ix).

[0035] Particularly preferred, the Botulinum neurotoxin is in the form of an aqueous formulation comprising Botulinum neurotoxin, sodium chloride and human serum albumin, or an aqueous formulation comprising Botulinum neurotoxin, sodium chloride, human serum albumin and lactose, or an aqueous formulation comprising Botulinum neurotoxin, sodium chloride, human serum albumin and sucrose. Another particularly preferred aqueous formulation comprises Botulinum neurotoxin, sodium chloride, human serum albumin and histidine. Such formulations are, for example, disclosed in WO 2023 / 156389 and WO 2023 / 156385. The Botulinum neurotoxin can be as defined herein above, in particular, the Botulinum neurotoxin is in a form that is free of complexing proteins or is in the form of a complex that contains complexing proteins, and preferably is Botulinum toxin type A in a form that is free of complexing proteins or Botulinum toxin type A in the form of a complex that contains complexing proteins.

[0036] The present invention further pertains to magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for use in the treatment of treatment of infantile cerebral palsy, cervical dystonia, spasticity, preferably spasticity of large muscle areas or groups, an overactive bladder, for stroke rehabilitation, such as for improvement of the motor function or for motor function recovery of extremities of stroke patients, reduction of muscle hypertension or stiffness and improvement of muscular flexibility, or for improvement of muscle function of patients with central paralysis, of chronic pain, migraine, or regeneration of spinal cord injury, preferably an overactive bladder, central paralysis, chronic pain, muscle hypertension or regeneration of spinal cord injuries, in a subject, preferably a human subject. Ine one preferred embodiment, the magnetic stimulation is repetitive transcutaneous magnetic stimulation, preferably spinal or peripheral magnetic stimulation. In a preferred embodiment, the administration is via intramusclular injection / administration. In another preferred embodiment, the magnetic stimulation is performed after Botulinum neurotoxin (BoNT) administration, preferably directly after BoNT administration, more preferably 1 second to 60 minutes after botulinum neurotoxin (BoNT) administration, even more preferably 1, 2, 3, 4, 5, 10, 20 or 30 second(s), 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes or 60 minutes after BoNT administration, or between 1 second and 10 minutes, or between 5 seconds and 5 minutes, or between 10 seconds and 2 minutes, after BoNT administration. In a further preferred embodiment, magnetic stimulation in combinationwith Botulinum neurotoxin administration causes a visible muscle contraction in the subject, preferably human subject. In a still further preferred embodiment, the Botulinum neurotoxin is BoNT / A, preferably NT201. In still another embodiment, the dose of BoNT / A, preferably NT201, is a dose of 1-24 U / kg, preferably 10-16 U / kg, more preferably 12-16 U / kg or 15-16 U / kg, even more preferably 16 U / kg body weight in human. As demonstrated in the following Examples, the use of magnetic stimulation in combination with BoNT / A administration resulted in an increase of the therapeutic effect / potency of BoNT / A, in comparison to administration of BoNT / A alone, in a mouse model. Preferably, said therapeutic effect / potency of BoNT in the treatment of a disease or disorder in a subject is increased at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold or even more fold, more preferably, between 1.1-fold and 1.5 fold, in comparison to administration of BoNT alone. In addition, magnetic stimulation in combination with BoNT administration can be used for extending the duration of action of Botulinum neurotoxin, i.e. said combination results in a longer duration of time for eliciting its efficient effect. Preferably, said longer duration of time is at least 30 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, one week, two weeks, three weeks, four weeks, six weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or even longer, or between 30 minutes and 12 months, between 1 hour and 11 months, between 12 hours and 10 months, between 24 hours and 9 months, or between 6 months and 9 months. The definitions, explanations, advantages and embodiments with respect to the uses of the invention apply mutatis mutandis to the medical uses of the invention.

[0037] The following Example provides an appropriate magnetic stimulation protocol for triggering muscle contractions by nerve stimulation in a mouse model, thereby enabling peripheral and spinal repetitive magnetic stimulation of nerves for increasing the therapeutic effects / potency of Botulinum neurotoxin in mouse. This study can be used for establishment of analogous stimulation protocols in human.

[0038] As used herein, the singular forms “a”, “an” and “the” include both singular and plural reference unless the context clearly dictates otherwise. By way of example, “a cell” refers to one or more than one cell.

[0039] As used herein, the term “about” when qualifying a value of a stated item, number, percentage, or term refers to a range of plus or minus 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent or 1 percent of the value of the stated item, number, percentage, or term. Preferred is a range of plus or minus 10 percent.

[0040] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonyms with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Evidently, the term “comprising” encompasses the term “consisting of’. More specifically, the term “comprise” as used herein means that the claim encompasses all the listed elements or method steps, but may also include additional, unnamed elements or method steps. For example, a method comprising steps a), b) and c) encompasses, in its narrowest sense, a method which consists of steps a), b) and c). The phrase "consisting of means that the composition (or device, or method) has the recited elements (or steps) and no more. In contrast, the term “comprises” can encompass also a method including further steps, e.g., steps d) and e), in addition to steps a), b) and c).

[0041] In case numerical ranges are used herein such as “in a concentration between 1 and 5 micromolar”, the range includes not only 1 and 5 micromolar, but also any numerical value in between 1 and 5 micromolar, for example, 2, 3 and 4 micromolar.

[0042] The term “ / / / vitro" denotes outside, or external to, the animal or human body. The term “ / / / vitro" as used herein should be understood to include “ex vivo” . The term “ex vivo” typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, e.g., in a culture vessel. The term “ / / / vivo” as used herein denotes inside, or internal to, the animal or human body.

[0043] As used herein, the term “magnetic stimulation” means the stimulation of a nerve, organ, or muscle etc. by means of a magnetic field, in its broadest sense. More specifically, it means a technique for stimulating peripheral nerves and cerebral cortex in order to help quantify the integrity of the motor nervous system, especially to measure conduction times. Its purpose is to create a pulsed electric current, induced by the time-varying magnetic field that will momentarily depolarise the nervous system. It is important to acknowledge that the actual pathways being investigated are not known; however, they incorporate the fastest conducting fibres which presumably include the pyramidal tracts. A magnetic field is generated by passing an electric current through a coil of wire, called the magnetic coil,which is placed e.g. above the scalp. Faraday's law says that whenever a magnetic field changes, there is an induced electric field which impedes the changing magnetic field. The magnetic pulse produced from an electric current pulse will thus induce in turn a current in an electrically conductive region, such as the human or animal neuronal axons. This induced electric current flows perpendicularly to the magnetic field and circulates up to a few centimetres away from the coil's external edge, and with a direction opposite to the current flowing in the coil and an intensity proportional to the magnetic field. The intensity of the magnetic field can be represented by flux lines around the coil and is measured in Tesla (T). The magnetic field is oriented perpendicular to the coil and, for currently available devices, can reach values of up to 7 T. The precise stimulating characteristics depend upon the model of stimulator used. For example, the Magstim 200 stimulator (Magstim) produces a magnetic field which rises to peak within about 150 microseconds and then decays slowly to zero over the next millisecond. Such a rapidly changing magnetic field induces electric eddy currents in any conductive structures nearby. Because the skull presents a low impedance to magnetic fields of this frequency, eddy currents are produced in the brain, and these currents can stimulate neural tissue. Currents induced on the scalp by magnetic stimulation are much weaker than those produced by transcranial electrical stimulation used in neuroscientific research because they crossed the extracerebral layers (scalp, skull and meninges) with minimal or no activation of the pain receptors and resulted in a well-tolerated procedure. Therefore, the sensation produced by magnetic stimulation is very slight. In a homogenous medium, the electric field will cause the current to flow in loops parallel to the plane of the coil. The loops with the strongest current will be near the circumference of the coil itself. The current loops become weak near the centre of the coil, and there is no current at the centre itself. The magnetic field decreases rapidly with increasing distance from the coil: with a typical 12 cm diameter round coil the strength falls by half at a distance of 4-5 cm from the coil surface. Since the cerebral cortex can be 1-2 cm from the surface of the scalp, and since the central sulcus itself can be 2 cm deep in man, this means that stimulation is severely attenuated at deep sites such as basal ganglia or thalamus. The magnetic motor evoked potential (MMEP) testing can be regarded as a counterpart of the longer-established procedure of somatosensory evoked potential (SSEP) monitoring, where small “cortical” potentials are recorded over the scalp in response to peripheral nerve stimulation. When magnetic stimulation is performed on the motor cortex, electromyographic responses (MMEPs, magnetic motor evoked potentials) can be recorded in contralateral, particularly distal, appendicular muscles. However, large pulses of magnetic field need to be generated in order to induce electric fields in the body of sufficient amplitude and duration to causestimulation of the neural tissue in its vicinity. Therefore, “magnetic stimulators” as used herein consist of a coil of wire connected to a large electrical capacitance. A magnetic stimulator works by charging one or more energy storage capacitors and then rapidly transferring this stored energy from the capacitor(s) to the stimulating coil as it discharges. Current (with a peak value of 5,000 A or more) flows thus from the capacitor through the stimulating coil generating the required magnetic field.

[0044] „Repetitive magnetic stimulation44as used herein uses magnetic stimulators that can deliver repetitive magnetic pulses to the subject to be treated, such as duplicate pulses, bursts, e.g. theta bursts, or series of pulses, e.g. low-frequency pulses or high-frequency pulses. “Repetitive transcranial magnetic stimulation” (rTMS) is a safe and non-invasive treatment technique used to treat, for instance, various psychiatric and neurological disorders. It involves the stimulation of specific deep brain regions by the production of high and low-intensity magnetic fields which modulates the cortical excitability. “Repetitive peripheral magnetic stimulation (rPMS)” describes a technique of non-invasive stimulation of nerves, muscles, spinal roots or even the autonomic nervous system: a coil positioned on the skin elicits high-frequency repetition of magnetic impulses.

[0045] The term “Botulinum neurotoxin (BoNT)” or “Botulinum neurotoxin (BoNT) polypeptide” as used herein comprises BoNT / A, BoNT / Al, BoNT / A2, B0NT / A6, BoNT / B, BoNT / Cl, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or XEOMIN® (IncobotulinumtoxinA: NT201; Merz Pharma GmbH & Co. KGaA).

[0046] The term “biological activity of a Botulinum neurotoxin (BoNT) polypeptide” means the biological properties characteristic for a Botulinum neurotoxin polypeptide, namely, a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and / or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle membrane fusion. It is envisaged that the Botulinum neurotoxin polypeptide exhibits at least one of the properties a) to d) mentioned above, preferably endoproteolytic cleavage of proteins involved in synaptic vesicle membrane fusion, or two or three or more preferably all four biological properties listed in a) to d).

[0047] The terms “administering” and “administration” refer to any method of providing a drug or pharmaceutical composition such as a Botulinum neurotoxin polypeptide to a subject. Such methods are well known to those skilled in the art and include, but are not limited to: oral administration, transdermal administration, administration by inhalation,nasal administration, topical administration, intravaginal administration, ophthalmic administration, intramural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. In various aspects, a drug or pharmaceutical composition such as a Botulinum neurotoxin polypeptide can be administered therapeutically; that is, administered to treat an existing disease or disorder or pathological condition in a subject as referred to herein. For instance, magnetic stimulation can be performed in combination botulinum neurotoxin (BoNT) administration for use in the treatment of a disease or disorder in a subject, according to the uses of the present invention. To this end, botulinum neurotoxin (BoNT) is administered to the subject e.g. by intramuscular injection, then the subject’s body is exposed to magnetic fields by magnetic stimulation. The skilled person or medical practitioner can determine an efficacious dose, an efficacious schedule, or an efficacious route of administration of a Botulinum neurotoxin polypeptide so as to treat a subject. “Potency” of a Botulinum neurotoxin as used herein is the intensity of effect produced for a given Botulinum neurotoxin dose. For instance, two drugs can be equi efficacious, i.e., produce the same maximal response, but vary in potency (dose required to produce the response). The drug that requires the larger dose to produce the desired effect is said to be less potent. A “therapeutic effect” of a Botulinum neurotoxin as used herein means the intended beneficial effects of the Botulinum neurotoxin in the treatment of a disease or disorder as referred to herein. For instance, the use of magnetic stimulation in combination with BoNT / A administration resulted in an increase of the therapeutic effect / potency of BoNT / A, in comparison to administration of BoNT / A alone, in a mouse model, as demonstrated in the following Examples. Preferably, said therapeutic effect / potency of BoNT in the treatment of a disease or disorder in a subject is increased at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold or even more fold, more preferably, between 1.1 -fold and 1.5 fold, in comparison to administration of BoNT alone. In addition, magnetic stimulation in combination with BoNT administration can be used for extending the duration of action of Botulinum neurotoxin, i.e. said combination results in a longer duration of time for eliciting its efficient effect. Preferably, said longer duration of time is at least 30 minutes, 45 minutes, 60 minutes, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, one week, two weeks, three weeks, four weeks, six weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or even longer,or between 30 minutes and 12 months, between 1 hour and 11 months, between 12 hours and 10 months, between 24 hours and 9 months, or between 6 months and 9 months.

[0048] The term “treatment” as used herein denotes the improvement or even elimination of one or more, preferably all symptoms associated with a disease or disorder as referred to herein by the administration of a pharmaceutical composition or drug or medicament such as a Botulinum neurotoxin polypeptide to a subject in need thereof. The term “treatment” as used herein can preferably encompass the improvement or even elimination of one or more, preferably all “side effect(s)”, “adverse effect(s)” “adverse reaction(s)” or “adverse event(s)” mentioned herein that are caused by administration of a Botulinum neurotoxin polypeptide, in particular high doses thereof, to a subject suffering from a disease or disorder referred to herein. Side effects or adverse effects are the negative side effects which are the unwanted effects of the Botulinum neurotoxin in the treatment of a disease or disorder as referred to herein. Such side effects encompass, for instance, muscle weakness, vision problems, trouble talking or swallowing, breathing problems, allergic reaction, or loss of bladder control.

[0049] The term “disease” broadly refers to any condition that impairs the normal functioning of the body of a human or animal. For this reason, diseases are associated with the dysfunction of the human or animal body's normal homeostatic processes. The term “disorder” means a functional abnormality or disturbance, in medicine. The term “pathological condition” means abnormal anatomical or physiological conditions and objective or subjective manifestations of disease, not classified as disease or syndrome.

[0050] The term „motor fimction“ can be defined as the ability to produce bodily movements when the brain, motor neurons, and muscles interact.

[0051] The term „muscle hypertonia“ or „hypertonia“ can be defined as abnormally and involuntarily increased resistance to externally imposed movement about a joint.

[0052] The term „muscle stiffness“ can be defined as the change in force divided by the corresponding change in length, when the length change is imposed by an external agent or by a change in the external load on the muscle. Stiffness is time-varying and therefore has dynamic attributes. Classically, stiffness refers only to the elastic, and therefore static, component of impedance. However, muscular properties are nonlinear, and therefore muscular impedance cannot easily be partitioned into components corresponding to thederivatives of position. Researchers in motor systems have chosen to retain the term “stiffness” to describe the resistance of muscle to length change.

[0053] The term „muscle flexibility^ refers to the ability of muscles to move through an unrestricted, pain-free range of motion. It involves the capacity of these structures to stretch, lengthen, and contract without limitations, allowing for smooth and efficient movement.

[0054] The term “subject” as used herein refers to an animal or human as set forth herein. The term does not denote a particular age or sex of the subject. Subject can be used interchangeably with “individual” or “patient” (if human).

[0055] As used herein, “nerves” or “nerve cells” or “neurons” are the fundamental units of the brain and nervous system, the cells responsible for receiving sensory input from the external world, for sending motor commands to our muscles, and for transforming and relaying the electrical signals at every step in between.

[0056] As used herein, the term “dose” means the amount of medicine or drug or pharmaceutical composition such as Botulinum neurotoxin taken, at one time.

[0057] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0058] The Figure shows:Figure 1: Impact of magnetic stimulation on the digit abduction score in mice. The combination with magnetic stimulation leads to an increase in the potency of the toxin from day 7 to day 25 in the higher dose groups. Two-way RM ANOVA followed by Bonferroni's multiple comparisons test *p < 0.05 on day 21 treated stimulated vs. nonstimulated for 30 U / kg. N = 10 animals per group. Data are shown as mean + / - SEM.Administration of 30 U / kg BoNT / A in combination with peripheral magnetic stimulation increased the potency in the DAS from day 7 to day 25 in comparison to the nonstimulated group reflecting a higher efficacy in the stimulated group.

[0059] The invention will now be illustrated by the following example which shall, however, not be construed as limiting the scope of the present invention.

[0060] Example 1: Investigating the influence of magnetic nerve stimulation on the paralytic effect of NT201 in miceThe aim of the study was the investigation of the effects of BoNT / A (NT201) combined with peripheral or spinal magnetic stimulation in mice.AnimalsAdult male Balb / c mice between 5-7 weeks old were purchased from Janvier Labs. Mice were kept on a 12 h light / dark cycle. All animals were maintained in an enriched environment under a constant temperature (22 ± 2°C) and humidity (30-70%) with food and water available ad libitum. Animals were acclimatized for at least 7 days prior to experimentation.Study designAfter the acclimatization period all mice were manually assigned into five groups with n=10 mice / group. All groups are summarized in Table 1.Table 1*Dose calculation referring to 20 g mouse body weightAt the first day of the experiment (day 0) mice of all groups were anesthetized with isoflurane 5% before i.m. injection of test or reference compound. Therefore, the right hindlimb was shorn with an electric clipper.Groups 1-5 were administered into the right hindlimb tibialis anterior muscle with the 0.4 LDU (groups 1 and 3) or 0.6 LDU (groups 2 and 4) NT201 or 0.2% HAS in 0.9% saline (group 5) using a Hamilton syringe with 30-gauge needle. The total volume for all injections was 20 pl.Within two minutes after BoNT / A injection, mice of groups 3-5 were magnetically stimulated.Magnetic stimulationMice were placed in a dorsal position on the coil. Thus, the skin in the mid body part of the mice was in direct contact with the coil which enabled the maximum magnetic stimulating of the spinal cord passing underneath as the highest magnetic field was produced by the coil in that area. Stimulation led to visible contractions of pelvic and hindlimb muscles.The stimulation was perfomed with 500 V at 2 Hz frequency. One Pulse train consisted of 30 stimuli followed by a pause of 50 s. The pulse train was started and stopped by the experimenter using a pedal coupled to the stimulator. The duration of one pulse train was 15 s. The pulse train was started 10 times (1 train: 15 s on, 50 s off).Digit abduction scoreThe local muscle weakening efficacy was determined in mice by the digit abduction score (DAS) in order to quantify the paralytic effect of botulinum toxin type A (see Aoki et al., 2001) after i.m. administration. The degree of abduction of the hind digits during theshortening reaction upon tail suspension was scored via a modified 0-4 scale which was subdivided into 0.25 scoring steps creating a 17-point scale. The method was established by the experimenter from preclinics. A score of 0 indicated normal digit abduction whereas a score of 4 indicated the maximal reduction in digit abduction (maximal muscle weakening).The mice were grabbed by the back fur and the abduction degree of the digits of the injected right hind paw was scored. The procedure was repeated 3 times before the animals were placed again in the respective polycarbonate cages. Out of the three detection procedures the lowest score per animal per time point were selected for further evaluation in order to detect the maximal muscle performance per animal.ParameterThe body weight as well as the DAS of all animals was detected regularly throughout the study. Both parameters were determined at baseline before test item injection (day minus 3), at 18 and 24 h after injection, at days 1,2, 3, 4 and after day 4 every third study day.At the end of the in-life period of 30 days animals were euthanized via cervical dislocation in deep isoflurane anesthesia.ResultsTable 2As shown in Figure 1 and Table 2, the combination with magnetic stimulation lead to an increase in the potency of BoNT / A from day 7 to day 25 in the higher dose groups. Specifically, administration of 30 U / kg BoNT / A in combination with peripheral magnetic stimulation increased the potency in the DAS from day 7 to day 25, in comparison to the non-stimulated group reflecting a higher efficacy / potency of BoNT / A in the stimulated group.

Claims

Claims1. Use of magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for increasing the therapeutic effect / potency of Botulinum neurotoxin (BoNT), in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT alone.

2. Use of magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for extending the duration of action of BoNT, in the treatment of a disease or disorder in a subject, in comparison to the treatment of said disease or disorder in said subject by BoNT administration alone.

3. Use of magnetic stimulation in combination with Botulinum neurotoxin (BoNT) administration for lowering the incidence of adverse effects of Botulinum neurotoxin (BoNT) in the treatment of a disease or disorder in a subject, compared to the treatment of said disease or disorder in said subject by BoNT administration alone.

4. Use of any one of claims 1 to 3, wherein magnetic stimulation is repetitive magnetic stimulation.

5. Use of claim 4, wherein magnetic stimulation is repetitive transcutaneous magnetic stimulation or repetitive transcranial magnetic stimulation.

6. Use of any one of claims 1 to 5, wherein magnetic stimulation is performed using one or more magnetic coil(s) connected to a magnetic stimulator.

7. Use of any one of claims 1 to 6, wherein the magnetic stimulation is performed after Botulinum neurotoxin (BoNT) administration, preferably after 1 second to 60 minutes.

8. Use of any one of claims 1 to 7, wherein the following parameter settings are used for the magnetic stimulation: a frequency of 0.1-50 Hz, and an intensity of 1-130%.

9. Use of any one of claims 1 to 8, wherein Botulinum neurotoxin administration is via intramuscular administration.

10. Use of any one of claims 1 to 9, wherein Botulinum neurotoxin (BoNT) is administered in a dose of 1-30 U / kg body weight in mouse, and a dose of 1-24 U / kg body weight in human.

11. Use of any one of claims 1 to 10, wherein the Botulinum neurotoxin is BoNT / A, BoNT / Al, BoNT / A2, B0NT / A6, BoNT / B, BoNT / Cl, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or XEOMIN® (IncobotulinumtoxinA: NT201), preferably BoNT / A or XEOMIN® (IncobotulinumtoxinA: NT201).

12. Use of any one of claims 1 to 11, wherein the subject is a mammal such as a mouse or human.

13. Use of any one of claims 1 to 12, wherein magnetic stimulation in combination with Botulinum neurotoxin administration causes a visible muscle contraction in the subject.

14. Use of any one of claims 1 to 13, which is for treatment of infantile cerebral palsy, cervical dystonia, spasticity, preferably spasticity of large muscle areas or groups, an overactive bladder, for stroke rehabilitation, such as for improvement of the motor function or for motor function recovery of extremities of stroke patients, reduction of muscle hypertension or stiffness and improvement of muscular flexibility, or for improvement of muscle function of patients with central paralysis, of chronic pain, migraine, or regeneration of spinal cord injury.

15. Use of claim 14, which is for treatment of an overactive bladder, central paralysis, chronic pain, muscle hypertension or regeneration of spinal cord injuries.

Images