Composition modulating botulinum neurotoxin effect

Abstract

To provide a method for modulating the effect of a botulinum neurotoxin composition, that is accelerating the onset of action of the botulinum neurotoxin composition and / or extending the duration of the action and / or enhancing the intensity of the action, and to provide a composition suitable for implementing the method.SOLUTION: Provided is a method comprising a step of adding at least one postsynaptic inhibitor of cholinergic neuronal transmission to a botulinum neurotoxin composition. Also provided are a composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin and uses thereof for treating aesthetic or therapeutic conditions.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This patent application claims priority to European Patent Application No. 19181635.4, filed June 21, 2019, the contents of which are incorporated herein by reference.

[0002] The present invention relates to compositions comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin, and their use for various therapeutic and / or cosmetic purposes. Furthermore, the compositions and methods in which the compositions are used provide advantageous treatments that result in enhancing the effects of the botulinum neurotoxin, e.g., accelerating the onset of its action and / or prolonging the duration of its action and / or increasing the intensity of its action. [Background technology]

[0003] The structure of BoNT Botulinum neurotoxins are toxins that inhibit neuronal transmission, acting preferentially on cholinergic neurons. Therefore, one of the primary consequences of botulinum neurotoxin action is muscle relaxation due to reduced neuronal stimulation. Botulinum neurotoxins are produced as complexes by the anaerobic, spore-forming bacterium Clostridium botulinum and, to a lesser extent, by other Clostridium species, such as Clostridium butyricum, Clostridium barati, Clostridium sporogenes, and Clostridium argentinense. Eight distinct serotypes of botulinum neurotoxins have been identified, known as types A (BoNT / A), B (BoNT / B), C (BoNT / C), D (BoNT / D), E (BoNT / E), F (BoNT / F), G (BoNT / G), and X (BoNT / X). Currently, eight subtypes of BoNT / A, eight subtypes of BoNT / B, twelve subtypes of BoNT / E, and nine subtypes of BoNT / F are known, respectively. Types A, B, E, and F are toxic to humans, whereas types C, D, and G are often toxic to birds, horses, cattle, and primates, for example. Type X was only recently reported after a case of infant botulism was reported in Japan in 1995 (Zhang et al., Nature Communications, Vol. 8, 14130 (2017)).

[0004] The botulinum neurotoxin complex exists in the form of a high molecular weight protein complex containing two components: an enzymatically active neurotoxin component and related non-toxic bacterial protein components, which can be considered coat proteins, including hemagglutinin and non-hemagglutinin proteins. The molecular weight of the botulinum toxin complex varies from approximately 300 kDa to approximately 900 kDa depending on the botulinum toxin serotype, while the molecular weight of botulinum neurotoxins lacking the non-toxic bacterial protein components is approximately 150 kDa. For cosmetic or therapeutic applications, the coat protein has been reported to have no significant function and does not contribute to the neurotoxic properties. The neurotoxin component is expressed as an inactive single-chain precursor with a molecular weight of approximately 150 kDa for all known botulinum neurotoxin serotypes. This single-chain precursor is activated by proteolytic cleavage to generate a disulfide-linked two-chain protein. The approximately 50 kDa light chain protein contains the catalytic domain and is a zinc-containing metalloprotease that acts as a zinc endopeptidase. The approximately 100 kDa heavy chain protein contains the translocation domain and the receptor-binding domain. This heavy chain mediates binding to presynaptic cholinergic nerve terminals, particularly the motor end plate and the presynaptic portion of the neuromuscular junction, and internalization of the neurotoxin into the cell.

[0005] BoNT mechanism of action Upon reaching the effector tissue, usually the neuromuscular junction, the heavy chain of botulinum neurotoxin mediates preferential uptake by cholinergic neurons through dual binding to presynaptic receptors (synaptic vesicle 2 protein) and specific gangliosides. The botulinum neurotoxin then enters neurons via receptor-mediated endocytosis and resides within endocytic vesicles. Upon vesicle acidification, the light chain translocates into the cytoplasm and undergoes cleavage. Depending on the BoNT serotype, the toxic portion of the light chain can cleave one or more of the proteins that form the SNARE protein complex (i.e., SNAP-25, syntaxin, and VAMP). The SNARE complex typically enables membrane fusion between the vesicle and the cell membrane, thereby releasing the neurotransmitter acetylcholine. For example, the release of the neurotransmitter acetylcholine at the synaptic cleft transmits nerve impulses to muscles, signaling them to contract. For example, when BoNT / A cleaves the proteins that essentially form the SNARE complex, preventing the formation of the SNARE complex, it blocks exocytosis of neurotransmitters, particularly the release of acetylcholine. As a result, communication between nerves and muscles (or other effector tissues) is blocked. In particular, the catalytically active light chain of botulinum neurotoxin can remain in nerve cells for several weeks, thereby enabling long-term and reversible inhibition of neurotransmission.

[0006] Medical Use of BoNT Because botulinum neurotoxins have the natural ability to reversibly block acetylcholine release from presynaptic nerve terminals in muscles and other effector tissues, such as the glands of the sympathetic and parasympathetic nervous systems, they have become an important therapeutic option in many fields to control overstimulation of muscles (and other effector tissues) and the associated pain. For example, BoNT / A is currently used therapeutically in the field of movement disorders, particularly for the management of spasticity and dystonia; BoNT / A is well known for its effective relief of dystonia-related pain; and in the field of disorders of the urinary system, particularly for overactive bladder, and in the field of disorders of the secretory system, i.e., hyperhidrosis and sialorrhea. Furthermore, botulinum neurotoxins are currently used for various cosmetic indications, such as smoothing facial lines and reducing frown lines and periorbital wrinkles.

[0007] BoNT preparations Although botulinum neurotoxins share similar metalloprotease activity, involved in cleaving proteins of the SNARE complex, the onset and / or duration of action may differ among serotypes. For example, BoNT / A and BoNT / F induce complete localized paralysis of the hind limbs in mice within two days, whereas BoNT / E produces the same effect within 24 hours. Nevertheless, the duration of neuromuscular paralysis caused by BoNT / A is 28 days in mice, compared with only 5 and 8 days for toxins E and F, respectively. A similar pattern has been observed in humans, albeit on a different timescale. BoNT-A-induced paralysis is typically observed within one week and can last for 3–4 months (Davletov et al., TRENDS in Neurosciences 28 (2005); pp. 446–452). Conversely, BoNT-E exhibits a faster onset (24 hours) but a much shorter duration (less than 1 month) in humans (Yoelin et al., Plast Recontr Surg 142(2018); pp. 847e-855e).

[0008] Most attention has focused on BoNT / A due to its unique profile and activity in humans. Since 1991, several commercially available botulinum neurotoxin type A agents have been approved by the U.S. Food and Drug Administration (FDA). Available forms of BoNT / A include Botox® / Vistabel® (Allergan), Dysport® / Azzalure® (Ipsen Biopharm), and Xeomin® / Bocouture® (Merz Pharmaceuticals), among others. The only other BoNT serotype currently commercially available is BoNT / B Myobloc® / Neurobloc® (Solstice Neurosciences), although its medical / cosmetic uses are anecdotal. Currently, improving the properties of BoNT is an important goal to better meet medical needs.

[0009] Limiting properties of BoNT The onset, duration and potency of action of BoNT are important properties that determine its usefulness as a pharmaceutical.

[0010] Onset of action In cosmetic indications, wrinkle reduction is generally observed 2–3 days after injection of BoNT / A, but complete blockage (e.g., prevention of the appearance of frown lines) takes approximately 2–3 weeks. A faster onset is often desired by treatment subjects. Similarly, in therapeutic applications of BoNT / A, such as the treatment of muscle disorders, a faster onset of BoNT action would clearly be beneficial to the patient. From the practitioner's perspective, a faster onset of muscle paralysis would facilitate easier patient monitoring. Ideally, injection and monitoring should occur on the same day.

[0011] Duration of action It is desirable to extend the duration of action of BoNT to allow for greater time between injections while maintaining efficacy. This is to limit the number of injections, which can be physically and financially burdensome for patients. It is also to limit the risk of developing neutralizing antibodies to BoNT, which could reduce the effectiveness of further BoNT treatments and thereby limit the subject's cosmetic or therapeutic options. While this avoids repeated injections of BoNT at short intervals, this can result in periods during which BoNT is not acting on the subject, potentially creating problems with patient compliance.

[0012] Strength of action Increasing the potency of BoNT's action could improve cosmetic outcomes and alleviate disorders and painful conditions. Nevertheless, increasing the injected dose of BoNT can lead to diffusion of BoNT away from the injection site, potentially causing systemic toxicity. It would be beneficial to have a way to increase the local efficacy of BoNT without increasing systemic toxicity commensurate with its local efficacy.

[0013] Need for improvement Therefore, there is a need for compositions that make it possible to overcome these shortcomings, and in particular for compositions containing botulinum neurotoxins that exhibit a faster onset of action and / or a longer duration of action and / or an increased potency of action of the botulinum neurotoxin.

[0014] Enhancement of BoNT effect BoNT molecule Due to the limitations of naturally occurring BoNTs, the first strategy to enhance the efficacy of these neurotoxins is to modify the protein itself. Current recombinant technologies offer the possibility of optimizing BoNT properties by introducing modifications into the BoNT sequence. The primary approach is to create chimeric neurotoxins by combining heavy and light chains from different BoNTs. Numerous attempts over the past few decades have demonstrated that it is indeed possible to modulate the effects of BoNTs. However, the optimized properties of chimeras often approximate those of one of the parent BoNTs. For example, Wang et al. (J Biol Chem. 2008, June 20;283(25):16993-7002) described chimeric neurotoxins made from BoNT / A and BoNT / E. Notably, the AE chimera (comprising a light chain from BoNT / A and a heavy chain from BoNT / E) behaved similarly to BoNT / A, albeit with a slightly enhanced potency and prolonged duration of action. Conversely, EA chimeras (containing the light chain from BoNT / E and the heavy chain from BoNT / A) behave similarly to BoNT / E.

[0015] Furthermore, another approach aims to combine BoNT / A with BoNT / B to broaden the range of tissues that can be targeted by the neurotoxin. A recent paper (Wang et al., Biochem J. 2012 May 15;444(1):59-67) and an international application (WO 2017 / 191315 A1) describe such chimeras.

[0016] In conclusion, previous attempts to engineer BoNT proteins have proven to only slightly modulate BoNT properties, and alternative approaches are therefore being explored.

[0017] Delivery of BoNT For BoNT to be effective, it must first reach its target cells, i.e., cholinergic neurons. Therefore, shortening the time it takes for BoNT to reach target tissues or increasing the duration and amount of BoNT present at the synaptic level after administration are attractive strategies for modulating BoNT effects. Such an approach has been explored by Revance Therapeutics, which has developed a formulation of BoNT / A containing a peptide related to the cell-penetrating peptide family. Several patents and patent applications describe this formulation (i.e., WO 2002 / 07773 or WO 2005 / 120546). Surprisingly, experiments in animal models suggest that this formulation appears to limit BoNT / A diffusion and enhance its stability (Stone et al., Toxicon. 2011 Aug;58(2):159-67).

[0018] The clinical trial aimed to demonstrate the improved efficacy of this formulation compared with unformulated BoNT / A without the peptide. However, the researchers used a higher dose of the formulation than unformulated BoNT / A (Carruthers et al., Dermatol Surg. 2017 Nov;43(11):1321-1331). Therefore, the increased duration of action observed with the formulation may be due solely to the increased dose.

[0019] Finally, a recent review (Hallett, M., Toxicon. 2015 Dec 1;107(0 0):64-67) emphasizes the low influence of diffusion on BoNT uptake. Current observations suggest that BoNT delivery to target tissues (after injection) is largely convection-driven.

[0020] In conclusion, strategies to modulate BoNT delivery to enhance its efficacy may have limited potential, and alternative approaches are still needed to achieve this goal.

[0021] BoNT biology Another strategy to enhance the effect of BoNT might be to use other molecules that interact with the mechanism of action of BoNT, such as entry of BoNT into neurons or translocation of the light chain into the cytosol of neurons.

[0022] Because different BoNTs share some common mechanisms of action, it has been tempting to combine BoNT subtypes with different characteristics, for example, combining the faster onset of action of BoNT / E with the longer duration of action of BoNT / A, to achieve at least additive effects. In particular, preclinical studies (Meunier et al., Molecular and Cellular Neuroscience 22 (2003); pp. 454–466) have shown that co-injection of BoNT / A and BoNT / E induces i) a short onset of action (similar to BoNT / E alone) and ii) a short duration of action (similar to BoNT / E alone). Similar results have been obtained in humans using BoNT injections into the extensor digitorum brevis (EDB) muscle of the foot. Surprisingly, EDB muscles double-injected with BoNT / A and BoNT / E recovered function with a time course similar to that of BoNT / E-injected muscles (Eleopra et al., Neuroscience Letters 256 (1998); pp. 135–138). All these results indicate that combining two BoNTs does not produce the effect expected from simply adding the effects of each BoNT administered independently. It is now understood that certain BoNTs have dominant effects compared to others, which precludes the use of additive BoNT combinations. Therefore, to improve the pharmacological profile of the currently widely used BoNT / A, a mixture of BoNTs is unlikely to be a therapeutic candidate.

[0023] Other candidates for interacting with BoNT's mechanism of action are neurotoxins that target the same presynaptic regions as BoNT. These toxins, like those from snakes and spiders, exist in the animal kingdom and can reversibly paralyze motor axon terminals, producing results similar to those observed with BoNT. However, their magnitude and mechanism of action are quite different, as are the onset and duration of neuromuscular junction blockade. In general, unlike BoNT, these presynaptic neurotoxins induce acute and rapid degeneration of motor axon terminals, followed by rapid recovery. Again, it is tempting to imagine the therapeutic potential of combining such presynaptic neurotoxins with BoNT. Surprisingly, when injected into muscles previously paralyzed with BoNT / A or BoNT / B, the presynaptic neurotoxins promote the restoration of neurotransmission (Duregotti et al., Toxins 7 (2015), pp. 5322–5336).

[0024] Other candidates for interacting with BoNT's mechanism of action include molecules that modulate the activity of presynaptic neurons. Notably, Thyagarajan et al. (J Pharmacol Exp Ther. 2009 Nov;331(2):361-71) reported that activation of the TRPV1 channel appears to inhibit BoNT entry into presynaptic neurons, thereby preventing BoNT's muscle relaxant effects. TRPV1 is a calcium channel expressed by presynaptic neurons, and in this study, TRPV1 was activated by the use of capsaicin. While the underlying mechanism is intriguing, the observed effect was limited to inhibition of BoNT's effects; no molecules that enhance BoNT's effects have been described.

[0025] Taken together, these results indicate that the therapeutic potential of combining BoNT with presynaptically active molecules is very limited.

[0026] Interestingly, there have been no previous reports of combining BoNT with molecules acting at the postsynaptic level to enhance its effects. [Prior art documents] [Patent documents]

[0027] [Patent Document 1] International Publication No. 2017 / 191315A1 Brochure [Patent Document 2] International Publication No. 2002 / 07773 Pamphlet [Patent Document 3] International Publication No. 2005 / 120546 Pamphlet [Non-patent literature]

[0028] [Non-Patent Document 1] Zhang et al., Nature Communications, Vol. 8, 14130 (2017) [Non-patent document 2] Davletov et al., TRENDS in Neurosciences 28(2005); pp. 446-452 [Non-patent document 3] Yoelin et al., Plast Recontr Surg 142(2018); pp. 847e-855e [Non-patent document 4] Wang et al., J Biol Chem. 2008 Jun 20;283(25):16993-7002 [Non-patent document 5] Wang et al., Biochem J. 2012 May 15;444(1):59-67 [Non-patent document 6] Stone et al., Toxicon. 2011 Aug;58(2):159-67 [Non-Patent Document 7] Carruthers et al., Dermatol Surg. 2017 Nov;43(11):1321-1331 [Non-patent document 8] Hallett, M., Toxicon. 2015 Dec 1;107(0 0):64-67 [Non-Patent Document 9] Meunier et al., Molecular and Cellular Neuroscience 22(2003); pp. 454-466 [Non-Patent Document 10] Eleopra et al., Neuroscience Letters 256 (1998); pp. 135-138 [Non-Patent Document 11] Duregotti et al., Toxins 7 (2015), pp. 5322-5336 [Non-Patent Document 12] Thyagarajan et al., J Pharmacol Exp Ther. 2009 Nov;331(2):361-71 Summary of the Invention [Means for solving the problem]

[0029] The inventors have now identified a way to enhance the effects of BoNT by combining it with a postsynaptic inhibitor of neuronal transmission (PoNT) that targets i) skeletal muscle, ii) smooth muscle, iii) cardiac muscle, and iv) secretory glands.

[0030] As a result, the present invention relates to a method for enhancing the effect of a botulinum neurotoxin composition, comprising adding at least one postsynaptic inhibitor of cholinergic neuronal transmission to the botulinum neurotoxin composition.

[0031] The efficacy of a botulinum neurotoxin composition can be enhanced by accelerating the onset of action (start of action) of the botulinum neurotoxin and / or by increasing the duration of action and / or by increasing the intensity of action.

[0032] In another aspect, the present invention provides a composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission (PoNT) and a botulinum neurotoxin (BoNT).

[0033] The PoNTs used in the compositions of the present invention are small postsynaptic molecules and / or peptides that specifically bind to postsynaptic receptors expressed by effector tissues such as muscle or glands.

[0034] The botulinum neurotoxin used in the compositions of the present invention is a botulinum neurotoxin type A, B, or E, or a combination of the heavy and light chains of a botulinum neurotoxin type A, B, or E.

[0035] The postsynaptic receptors expressed by skeletal muscle cells include or are selected from the group consisting of (α1)2β1δε nAChr, (α1)2β1δγ nAChr, RyR1, CaV1.1 and NaV1.4.

[0036] The postsynaptic receptors expressed by smooth muscle cells are selected from the group comprising or consisting of M3 mAChr, RyR2, CaV1.2 and NaV1.5.

[0037] The postsynaptic receptors expressed by cardiomyocytes include or are selected from the group consisting of M2 mAChr, RyR2, CaV1.1, CaV1.2 and NaV1.5.

[0038] The postsynaptic receptors expressed by the secretory cells are selected from the group comprising or consisting of M1 mAChR, M3 mAChR, alpha 1 adrenergic receptors and beta 1 adrenergic receptors.

[0039] The compositions of the present invention not only combine the rapid onset of action of PoNT with the extended duration of action of BoNT, but also PoNT synergistically enhances the onset, duration, and potency of action of BoNT.

[0040] In all embodiments, the at least one postsynaptic inhibitor of cholinergic neuronal transmission is compatible with the delayed action (slow onset) of the botulinum neurotoxin.

[0041] In certain embodiments, the at least one postsynaptic inhibitor of cholinergic neuronal transmission is a fast-acting postsynaptic peptide and / or a fast-acting postsynaptic small molecule.

[0042] The compositions of the present invention can further be used in cosmetic treatments, such as reducing wrinkles, lines such as glabellar lines, or furrows, muscle volume for cosmetic purposes (such as the masseter or calf muscles), hypertrophic scars, and other dermatological conditions (skin conditions).

[0043] The compositions of the present invention can further be used for therapeutic treatment, such as, for example, (i) musculoskeletal disorders including movement disorders, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the upper limbs, blepharospasm, eyelid disorders, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasm, hemifacial spasm, tremor, tics, teeth grinding (bruxism), apraxia, and freezing of gait, and (ii) treatment of pain associated with these disorders.

[0044] The compositions of the present invention can further be used to treat (i) smooth muscle disorders, including spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophageal disorders (esophageal disorders), gastroparesis, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, dysuria, fecal incontinence, constipation, anismus, anal fissures, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus (priapism), other muscle tone disorders and other disorders characterized by involuntary movement of muscle groups, and (ii) pain associated with these disorders.

[0045] The compositions of the present invention can further be used to treat cardiac disorders, including atrial fibrillation.

[0046] The compositions of the present invention can further be used to treat glandular disorders, including lacrimation, hyperhidrosis (hands, feet, and underarms), sialorrhea, excessive salivation, excessive gastrointestinal secretions, excessive sebaceous gland production (and related conditions such as acne), and secretory disorders.

[0047] In all embodiments, the PoNT and BoNT must be administered in combination, i.e., simultaneously or within a time schedule that allows for their simultaneous effective presence at the synaptic level. In other words, at least one postsynaptic inhibitor of cholinergic neuronal transmission (PoNT) and the botulinum neurotoxin may be administered simultaneously, separately, or at different times, provided that the botulinum neurotoxin is administered before the decay of PoNT activity occurs and, optionally, after the onset of PoNT activity.

[0048] Finally, the present invention relates to a kit for carrying out the method of the present invention, comprising a needle and corresponding syringe, at least one postsynaptic inhibitor of cholinergic neuronal transmission, and a botulinum neurotoxin.

[0049] The invention and its preferred embodiments are described in further detail below. [Brief explanation of the drawings]

[0050] [Figure 1] DAS assay: Groups of five rats were injected into the right tibialis anterior muscle with 5 U / kg BoNT / A or 5 U / kg BoNT / A combined with increasing doses ranging from 0.4 to 40 μg / kg of μ-conotoxin CnIIIc peptide and assessed for digit abduction scores (DAS) for up to 10 days. The mean DAS and standard error of the mean for each group at each time point are shown. Rats injected with saline into the left tibialis anterior muscle as a control had a DAS of 0 throughout the study period, and data are not shown in the graph. [Figure 2]DAS assay: Groups of five rats were injected into the right tibialis anterior muscle with 5 U / kg BoNT / A, 80 μg / kg μ-conotoxin CnIIIc, or a combination of 5 U / kg BoNT / A and 80 μg / kg μ-conotoxin CnIIIc and assessed for DAS for up to 12 days. Data from the first two conditions (5 U / kg BoNT / A, 80 μg / kg μ-conotoxin CnIIIc) were then summed and plotted ("Sum"). Rats injected with saline into the left tibialis anterior muscle as a control had a DAS of 0 throughout the entire study period, and data are not shown in the graph. [Figure 3] DAS assay: Groups of rats were injected into the right tibialis anterior muscle with 5 U / kg BoNT / A, 5 U / kg BoNT / A combined with 57 μg / kg pancuronium, 5 U / kg BoNT / A combined with 8 μg / kg α-conotoxin MI peptide, or 5 U / kg BoNT / A combined with 17 μg / kg α-bungarotoxin peptide, and assessed for DAS for up to 12 days. Rats injected with saline into the left tibialis anterior muscle as a control had a DAS of 0 throughout the study period, and data are not shown in the graphs. [Figure 4] DAS assay: Groups of rats were injected into the right tibialis anterior muscle with 5 U / kg BoNT / A, 5 U / kg BoNT / A combined with 1.6 μg / kg dantrolene, or 5 U / kg BoNT / A combined with 36 μg / kg of the insecticidal toxin LaIT1, and DAS was assessed for up to 12 days. Rats injected with saline into the left tibialis anterior muscle as a control had a DAS of 0 throughout the study period, and data are not shown in the graphs. [Figure 5] The synergistic effect of combining 5 U / kg of BoNT / A with either 25 μg / kg of wild-type μ-conotoxin CnIIIc peptide (SEQ ID NO: 54) or 25 μg / kg of mutant μ-conotoxin CnIIIc peptide (SEQ ID NO: 95) was analyzed using calculations of the area under the curve (AUC) during the early, middle, and late phases of the effect. [Figure 6]DAS assay: Groups of five rats were injected into the right tibialis anterior muscle with 5 U / kg BoNT / A, 5 U / kg BoNT / A combined with 83 μg / kg amlodipine, or 5 U / kg BoNT / A combined with 83 μg / kg diltiazem and 83 μg / kg verapamil, and DAS was assessed for up to 14 days. Rats injected with saline into the left tibialis anterior muscle as a control had a DAS of 0 throughout the study period, and data are not shown in the graphs. [Figure 7] The synergistic effects of combining 5 U / kg BoNT / A with small molecules that specifically inhibit NAChR receptors (pancuronium at 56.9 μg / kg and suxamethonium at 83 μg / kg), RYR1 receptors (dantrolene at 14 μg / kg), or CaV1.1 receptors (amlodipine at 83 μg / kg, diltiazem at 83 μg / kg, and verapamil at 83 μg / kg) were analyzed using calculation of the sum of the area under the curve (AUC). [Figure 8]The study included 5 U / kg BoNT / A and peptides that specifically inhibit NAChR receptors (α-bungarotoxin 16.8 μg / kg, α-conotoxin MI 0.8 μg / kg, waglerin-1 197.9 μg / kg, and αC-conotoxin PrXA 30.9 μg / kg), peptides that specifically inhibit RYR1 receptors (LaIT1 toxin 36 μg / kg, imperacalcin 32.8 μg / kg), and peptides that specifically inhibit CaV1.1 receptors (U7-ctenitoxin Pn1b 2.2 μg / kg, ω-conotoxin TxVII 24.8 μg / kg, and U6-ctenitoxin Pn1 100.6 μg / kg). The synergistic effects of combinations with peptides specifically inhibiting the Nav1.4 receptor (U9-ctenitoxin Pn1a at 33.8 μg / kg, κ-ctenitoxin Pn1 at 39.8 μg / kg, and glacontryphan M at 12.8 μg / kg) or peptides specifically inhibiting the Nav1.4 receptor (μ-conotoxin CnIIIC at 326 μg / kg, μ-conotoxin GIIIb at 23 μg / kg, μ-thomitoxin Hme1a at 36.4 μg / kg, μ-conotoxin GvIIJ at 32.5 μg / kg, μ-O-conotoxin MfVIA at 286.6 μg / kg, and μ-thomitoxin Hme1b at 39.8 μg / kg) were analyzed using the sum of the area under the curve (AUC). DETAILED DESCRIPTION OF THE INVENTION

[0051] The nervous system sends signals through electrical impulses (stimuli) that travel along the length of a nerve until it reaches a junction with another cell. A neural junction, also called a synapse, is the site where an electrical nerve impulse is transmitted between two nerve cells (neurons) or between a neuron and a gland or muscle cell (effector). Neuronal transmission involves the release of synaptic vesicles containing neurotransmitters by a presynaptic cell into the synaptic space, followed by the uptake of the vesicle contents by postsynaptic receptors expressed by the postsynaptic cell.

[0052] composition In a first aspect, the present invention relates to a composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission (PoNT) and a botulinum neurotoxin (BoNT).

[0053] By "at least one" is intended a single postsynaptic inhibitor of cholinergic neuronal transmission or a combination (i.e., a mixture) of two, three, four, five or more postsynaptic inhibitors of cholinergic neuronal transmission.

[0054] "Postsynaptic inhibitors of cholinergic neuronal transmission (PoNT)" refer to compounds that can bind to receptors on postsynaptic cells and interfere with cholinergic neuronal transmission. By binding to neurotransmitter target receptors, ion channels, or voltage-gated receptors present on the surface of postsynaptic cells, postsynaptic inhibitors of cholinergic neuronal transmission can modulate the effects of neurotransmitter (i.e., acetylcholine) release.

[0055] For the purposes of the present invention, postsynaptic inhibitors of cholinergic neuronal transmission can be divided into two main groups: peptidic inhibitors, called postsynaptic peptides, and non-peptidic inhibitors, called postsynaptic small molecules.

[0056] Thus, in the compositions of the present invention, the at least one postsynaptic inhibitor of cholinergic neuronal transmission is a postsynaptic peptide and / or a postsynaptic small molecule.

[0057] Postsynaptic peptides The term "postsynaptic peptide" refers to a biologically active peptide that has specific affinity for cell surface receptors present at the postsynaptic level (typically in the membrane of the postsynaptic cell). The postsynaptic peptides used in the compositions of the present invention may be naturally occurring neurotoxins isolated from the venom of spiders, snakes, scorpions, sea anemones, marine snails (boulders), and other animals, or functional fragments / variants thereof, or synthetic peptides or derivatives thereof obtained by chemical synthesis. A "fragment or variant" of a postsynaptic peptide refers to any subsequence of a postsynaptic peptide that retains the same biological activity as the parent peptide. That is, a functional fragment / variant of a postsynaptic peptide can specifically bind to and block postsynaptic receptors expressed by cells involved in the same effector tissue or the same family of effector tissues as those bound and blocked by the native postsynaptic peptide from which the fragment or variant is derived. A variant of a postsynaptic peptide may have at least 70%, at least 75%, 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% sequence identity with a native postsynaptic peptide, provided that the variant retains the binding and blocking activity to the target receptor. A functional fragment of a postsynaptic peptide encompasses the shortest possible peptide sequence that retains the binding and blocking activity of the native postsynaptic peptide.

[0058] "Derivative of a postsynaptic peptide" refers to any modified postsynaptic peptide compared to its native counterpart. All terminal modifications (i.e., amidation (C-terminus), acylation (N-terminus)) and internal modifications (e.g., leucine- or tyrosine-based motifs) that can improve the stability or biological function of the overall postsynaptic peptide should be considered. More generally, modifications can be selected from the group including or consisting of phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and PEGylation.

[0059] To enhance the bioavailability of the postsynaptic peptides used in the compositions of the invention, the postsynaptic peptides used in the compositions of the invention may include one or more cationic modifications.

[0060] The postsynaptic peptides used in the compositions of the present invention may contain at least one amino acid modification selected from an amino acid substitution, an amino acid insertion, and an amino acid deletion.

[0061] In an amino acid substitution, an amino acid residue that is part of the sequence of a postsynaptic peptide is replaced with a different amino acid residue. The replacement amino acid residue may be one of the 20 standard amino acids known in the art. Substitutions may occur between amino acids that are considered charged (aspartic acid, glutamic acid, arginine, and lysine), uncharged polar amino acids (asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan), or uncharged hydrophobic amino acids (alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine).

[0062] Alternatively, the replacement amino acid in the amino acid substitution may be a non-standard amino acid (an amino acid not included in the standard set of 20). For example, the replacement amino acid may be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, lysine, the D-isomers of arginine and ornithine, 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine. Methods for introducing non-standard amino acids into peptides are known in the art. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues in postsynaptic peptides.

[0063] In an amino acid insertion, an additional amino acid residue (one not normally present) is incorporated into the amino acid sequence of the postsynaptic peptide, thus increasing the total number of amino acid residues in that sequence.

[0064] In an amino acid deletion, an amino acid residue is removed from the amino acid sequence of the postsynaptic peptide, thus reducing the total number of amino acid residues in that sequence.

[0065] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art.

[0066] Depending on their size, postsynaptic peptides contain 1 to 10 amino acid modifications (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 4, 3, 2, or 1 amino acid modifications).

[0067] The postsynaptic peptides of the present invention can also contain non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, 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.

[0068] Since naturally occurring postsynaptic peptides are often mixtures of dozens of neurotoxins, any conventional purification process is likely to be useful for isolating postsynaptic peptides with the desired binding activity.

[0069] Alternatively, if highly purified postsynaptic peptides are required, synthetically produced postsynaptic peptides may be used to obtain high purity levels.

[0070] In all embodiments, the postsynaptic peptides used in the compositions of the invention are peptide chains comprising no more than about 110 amino acids, preferably no more than 70 amino acids, preferably no more than 50 amino acids, preferably no more than 35 amino acids, more preferably no more than 25 amino acids. In no case should the postsynaptic peptide, unless it is a fragment, be less than 7 amino acids in length.

[0071] When a fragment of a postsynaptic peptide is used in the compositions of the invention, it can be 3, 4, 5, or 6 amino acids in length.

[0072] According to a more preferred embodiment, the size of the postsynaptic peptide used in the composition of the present invention is in the range of about 80 to 105 amino acids, preferably about 50 to 80 amino acids, preferably about 40 to 60 amino acids, and more preferably about 15 to 35 amino acids.

[0073] According to a more preferred embodiment, the postsynaptic peptide used in the composition of the invention comprises at least one disulfide bridge.

[0074] low molecule "Postsynaptic small molecule" refers to a biologically active, non-peptide chemical molecule that has specific affinity for cell surface receptors present at the postsynaptic level (typically in the membrane of postsynaptic cells). Postsynaptic small molecules differ from postsynaptic peptides in that they lack peptide bonds and generally have a molecular weight less than 1300 g / mol (mole). Preferably, the molecular weight of the postsynaptic small molecules used in the compositions of the present invention is approximately 80-1200 g / mol, preferably approximately 150-800 g / mol, and more preferably approximately 300-600 g / mol. These small molecules may be extracted from nature (typically plants) or synthesized. Small molecules can generally be classified into families or related compounds; such classifications are described in detail herein as necessary. It is understood that a variety of small molecules within a family is contemplated.

[0075] BoNT "Botulinum neurotoxin" (BoNT) refers to the eight different naturally occurring botulinum neurotoxins, known as types A, B, C, D, E, F, G, and X, as well as modified, recombinant, hybrid, and chimeric botulinum neurotoxins. The terms botulinum neurotoxin and botulinum toxin are equivalent and can be used interchangeably. As used herein, the term "botulinum toxin complex" or "toxin complex" refers to the approximately 150 kD botulinum toxin protein molecule (belonging to any of botulinum toxin serotypes A-G, or X) together with potentially associated endogenous nontoxin proteins (i.e., hemagglutinin and nontoxin nonhemagglutinin proteins produced by Clostridium botulinum). Note, however, that a botulinum toxin complex need not be derived from Clostridium botulinum as a single toxin complex. For example, a botulinum toxin or modified botulinum toxin can first be recombinantly prepared and then combined with a non-toxin protein. Recombinant botulinum toxin can also be purchased commercially (e.g., from List Biological Laboratories, Campbell, Calif.) and then combined with a non-toxin protein.

[0076] Mutations in the coding sequence of a botulinum toxin that introduce one or more amino acid substitutions are referred to as modified BoNTs. A "modified botulinum neurotoxin" refers to a compound that has botulinum toxin activity but contains one or more chemical or functional changes in any portion or amino acid chain compared to a natural botulinum toxin or a recombinant, native botulinum toxin. For example, a botulinum toxin may be a modified neurotoxin in which at least one amino acid has been deleted, modified, or substituted compared to the native form. For example, a botulinum toxin may be modified in a way that enhances its properties or reduces undesirable side effects, while still retaining the desired botulinum toxin activity. A botulinum toxin may also be part of an entire molecule shown to have the required botulinum toxin activity, and in such cases may be used by itself or as part of a fusion protein combination or combined molecule, for example. In this case, the portion or fragment of a botulinum neurotoxin may be, for example, the 50 kDa light chain (LC) of the toxin. Alternatively, the botulinum toxin may be in the form of a botulinum toxin precursor, which may itself be non-toxic, for example, a non-toxic zinc protease that becomes toxic upon proteolytic cleavage.

[0077] "Hybrid and chimeric" BoNTs refer to mixtures of heavy and light chain domains of BoNTs of different serotypes or different subtypes. Hybrids and chimeras within a BoNT serotype and subtype may be naturally occurring, such as BoNT / FA and BoNT / CD, or may be recombinant variants of BoNT. Recombinant botulinum neurotoxins can have their light and / or heavy chains recombinantly produced by non-clostridial species.

[0078] According to a preferred embodiment, the botulinum neurotoxin used in the compositions of the present invention is of type A, type B, type E, or a combination of the heavy and light chains of types A, B, and E botulinum neurotoxins.

[0079] According to a more preferred embodiment, the botulinum neurotoxin used in the compositions of the present invention is type A.

[0080] Various effector tissues In all embodiments, the choice of at least one postsynaptic inhibitor of cholinergic neuronal transmission depends on the type of synapse, and more particularly on the postsynaptic cell involved in this synapse.

[0081] muscle The synaptic connection between a motor neuron and a muscle cell is called the neuromuscular junction (NMJ). At the NMJ, the postsynaptic cell can be a skeletal muscle cell, a smooth muscle cell, or a cardiac muscle cell.

[0082] skeletal muscle cells In one embodiment, when the postsynaptic cell is a skeletal muscle cell, the at least one postsynaptic inhibitor of cholinergic neuronal transmission used in the compositions of the present invention binds to at least one receptor expressed by the skeletal muscle cell, the receptor comprising or selected from the group consisting of a nicotinic acetylcholine receptor (nAChR), more preferably an (α1)2β1δε or (α1)2β1δγ nAChR, ryanodine receptor type 1 (RYR1), a voltage-gated L-type calcium channel CaV1.1, a voltage-gated L-type calcium channel CaV1.1, and a voltage-gated sodium channel NaV1.4.

[0083] When two or more postsynaptic inhibitors of cholinergic neuronal transmission (PoNTs) are used as components of the composition of the present invention, the receptors expressed by skeletal cells and targeted by these PoNTs may belong to the same receptor family or to several different receptor families.

[0084] Low molecular weight (skeletal muscle) In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule, the synaptic inhibitor of neuronal transmission is selected according to the target postsynaptic receptor expressed by the skeletal muscle cell, as shown in Table 1.

[0085] [Table 1(1)] [Table 1(2)]

[0086] Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels mediating fast synaptic transmission. Mammals have 16 nAChR subunits: nine α subunits (α1-7, α9, and α10), four β subunits (β1-4), and γ, δ, and ε subunits. Five of these subunits combine to form muscle nAChR subtypes (α1β1γδ and α1β1δε) present at the neuromuscular junction.

[0087] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to (α1)2β1δε or (α1)2β1δγ nAChR, the synaptic inhibitor of neuronal transmission is selected from the group comprising or consisting of rapacuronium, mivacurium, atracurium, doxacurium, cisatracurium, vecuronium, rocuronium, pancuronium, D-tubocurarine, pipecuronium, suxamethonium, decamethonium, or combinations thereof.

[0088] Any other small molecule known in the art that binds to (α1)2β1δε or (α1)2β1δγ nAChRs may be used as a synaptic inhibitor of one type of neuronal transmission in the compositions of the present invention.

[0089] In a more preferred embodiment, when the target receptor expressed by skeletal muscle cells is an (α1)2β1δε or (α1)2β1δγ nAChR, the at least one postsynaptic small molecule used in the compositions of the invention is pancuronium.

[0090] Ryanodine receptor isoform-1 (RyR1) is the major calcium channel in skeletal muscle, which is important for excitation-contraction coupling. It is a six-spanning homotetrameric protein present in the sarcoplasmic reticulum (SR) and transports Ca from the SR. 2+ Its function is to release ATP, which causes skeletal muscle contraction.

[0091] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to RyR1, the synaptic inhibitor of neuronal transmission is selected from the group including or consisting of dantrolene, azumolene, or a combination thereof.

[0092] In a more preferred embodiment, when the target receptor expressed by skeletal muscle cells is RyR1, the at least one postsynaptic small molecule used in the compositions of the present invention is dantrolene.

[0093] The voltage-gated L-type calcium channel CaV1.1 is found in muscle transverse tubules. In skeletal muscle, it binds to the ryanodine receptor RyR1 in the sarcoplasmic reticulum via a mechanical coupling. Upon depolarization of the skeletal muscle membrane, CaV1.1 undergoes a conformational change, allosterically activating RyR1.

[0094] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to CaV1.1, the synaptic inhibitor of neuronal transmission comprises or is selected from the group consisting of dihydropyridine, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, elgodipine, felodipine, flordipine, iganidipine, isradipine, lacidipine, lercanidipine, levumlodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, riodipine, verapamil, gallopamil, dimeditiapramine, diltiazem, or a combination thereof.

[0095] In a more preferred embodiment, when the target receptor expressed by skeletal muscle cells is CaV1.1, the at least one postsynaptic small molecule used in the compositions of the present invention is verapamil.

[0096] Postsynaptic peptides (skeletal muscle) In this embodiment, when the at least one postsynaptic inhibitor of cholinergic neuronal transmission is a postsynaptic peptide, the postsynaptic inhibitor of cholinergic neuronal transmission is selected according to the target postsynaptic receptor expressed by the skeletal muscle cell, as shown in Table 2.

[0097] [Table 2(1)] [Table 2(2)]

[0098] Postsynaptic peptides capable of binding to nAChRs (α1β1γδ and α1β1δε) and suitable for the compositions of the invention may be selected from the group comprising or consisting of azemiopsin (SEQ ID NO: 1), waglerin-1 (SEQ ID NO: 2 or SEQ ID NO: 3), SYN®-AKE (H-bAla-Pro-Dab-NHBn.2CH3CO2H), α-bungarotoxin (SEQ ID NO: 4), αC-conotoxin PrXA (SEQ ID NO: 5), α-cobratoxin (SEQ ID NO: 6), α-conotoxin MI (SEQ ID NO: 7 or SEQ ID NO: 8), α-conotoxin GI (SEQ ID NO: 7 or SEQ ID NO: 9), ψ-conotoxin-PrIIIE (SEQ ID NO: 10), candoxin (SEQ ID NO: 11 or SEQ ID NO: 12) and haditoxin (SEQ ID NO: 13 or SEQ ID NO: 14), or derivatives thereof.

[0099] The consensus sequence of waglerin-1 (SEQ ID NO: 2) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P24335, P58930, P24335, P58930.

[0100] In a preferred embodiment, when waglerin-1 is used in the composition of the present invention, the waglerin-1 has the sequence of SEQ ID NO: 3, or a derivative thereof.

[0101] The consensus sequence of α-conotoxin MI and α-conotoxin GI (SEQ ID NO: 7) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P01519, P01519, X5I9Y2, P01520, P0C8U4, P0DKP5, P0DKP8, P56973, P01521, P56973, P0C8U5, P0C8U4, P0C1W1, P0CAQ4, P0CAQ5, P56973, D4HPF8, D4HPE4, P0C8U5, P0C1W2, D4HPG9, D4HPF2, P0C8V1, P28878, P15471, P0DKP7, D4HPF6, P0DKP7, P28879.

[0102] In a preferred embodiment, when α-conotoxin MI or α-conotoxin GI is used in the compositions of the present invention, the α-conotoxin MI or α-conotoxin GI has the sequence of SEQ ID NO: 8, SEQ ID NO: 9, or a derivative thereof.

[0103] The consensus sequence of candoxin (SEQ ID NO: 11) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P81783, P15818, D5J9P6, D5J9P8.

[0104] In a preferred embodiment, when candoxin is used in the compositions of the present invention, the candoxin has the sequence of SEQ ID NO: 12, or a derivative thereof.

[0105] The consensus sequence of Hajitoxin (SEQ ID NO: 13) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: A8N286, Q9W727, P82464, D5J9P3, Q7ZT13, Q800Y3, Q9YGI0.

[0106] In a preferred embodiment, when a hargitoxin is used in the compositions of the present invention, the hargitoxin has the sequence of SEQ ID NO: 14, or a derivative thereof.

[0107] In an even more preferred embodiment, when the target postsynaptic receptor expressed by skeletal muscle cells is an (α1)2β1δε or (α1)2β1δγ nAChR, the at least one postsynaptic peptide used in the compositions of the invention is α-conotoxin MI having SEQ ID NO: 7, SEQ ID NO: 8, or a derivative thereof.

[0108] Postsynaptic peptides capable of binding to RyR1 and suitable for the compositions of the invention may be selected from the group comprising or consisting of imperacarcin (SEQ ID NO: 15 or SEQ ID NO: 16), phospholipase A2 imperatoxin-1 (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 18), heteromutoxin (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 19), phospholipase A2 (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 20), phospholipase A2 (SEQ ID NO: 17 or SEQ ID NO: 21), insecticidal toxin LaIT1 (SEQ ID NO: 22 or SEQ ID NO: 23) and φ-liotoxin LW1a (SEQ ID NO: 22 or SEQ ID NO: 24), or derivatives thereof.

[0109] The consensus sequence of Impellacalcin (SEQ ID NO: 15) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P59868, P60252, P60253, A0A1L4BJ42, P60254, P0DPT1, B8QG00, P0DM30, A0A1B3IJ19, L0GBR1, A0A1B3IJ24, A0A1W7RAU1, A0A224X3X5, A0A224X3Z6, A0A1V1WBN6.

[0110] In a preferred embodiment, when impellacalcin is used in the compositions of the present invention, the impellacalcin has the sequence of SEQ ID NO: 16, or a derivative thereof.

[0111] The phospholipase A2 imperatoxin-1 (large subunit), heteromutoxin (large subunit), phospholipase A2 (large subunit), and phospholipase A2 consensus sequences (SEQ ID NO: 17) have been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: Q6T178, Q3YAU5, P59888, H2CYP4, P0DMI6, A0A1L4BJ57, A0A1W7R9Y9, P0C8L9, A0A1W7RA04, A0A1L4BJ64.

[0112] In a preferred embodiment, when phospholipase A2 imperatoxin-1 (large subunit), or heteromutoxin (large subunit), or phospholipase A2 (large subunit), or phospholipase A2 is used in the compositions of the present invention, the phospholipase A2 imperatoxin-1 (large subunit), or heteromutoxin (large subunit), or phospholipase A2 (large subunit), or phospholipase A2 has the sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or a derivative thereof.

[0113] The consensus sequence of the insecticidal toxin LaIT1 and φ-liotoxin LW1a (SEQ ID NO: 22) has been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P0C5F2, P0DJ08.

[0114] In a preferred embodiment, when the insecticidal toxin LaIT1 or φ-liotoxin LW1a is used in the composition of the present invention, the insecticidal toxin LaIT1 or φ-liotoxin LW1a has the sequence of SEQ ID NO: 23, SEQ ID NO: 24, or a derivative thereof.

[0115] In an even more preferred embodiment, when the target receptor expressed by skeletal muscle cells is RyR1, the at least one postsynaptic peptide used in the compositions of the present invention is the insecticidal toxin LaIT1 having SEQ ID NO: 22, SEQ ID NO: 23, or a derivative thereof.

[0116] Postsynaptic peptides capable of binding to CaV1.1 and suitable for the compositions of the invention include calciceptin (SEQ ID NO: 25 or SEQ ID NO: 26), toxin FS2 (SEQ ID NO: 25 or SEQ ID NO: 27), vasotab (SEQ ID NO: 28 or SEQ ID NO: 29), glacontryphan M (SEQ ID NO: 30 or SEQ ID NO: 31), ω-conotoxin-TxVII (SEQ ID NO: 32), ω-ctenitoxin-Cs1a (SEQ ID NO: 33 or SEQ ID NO: 34), U7-ctenitoxin-Pn1b (SEQ ID NO: 35), U The toxin may be selected from the group comprising or consisting of 9-ctenitoxin-Pn1a (SEQ ID NO: 36 or SEQ ID NO: 37), U6-ctenitoxin-Pn1a (SEQ ID NO: 36 or SEQ ID NO: 38), κ-ctenitoxin-Pn1a (SEQ ID NO: 36 or SEQ ID NO: 39), ω-ctenitoxin-Pr2a (SEQ ID NO: 40 or SEQ ID NO: 41), toxin S4C8 (SEQ ID NO: 42 or SEQ ID NO: 43) and toxin C10S2C2 (SEQ ID NO: 42 or SEQ ID NO: 44), or a derivative thereof.

[0117] The consensus sequence of calciceptin and the toxin FS2 (SEQ ID NO: 25) has been defined on the basis of the sequences of proteins with the following accession numbers in the Uniprot database: P22947, P01414, P25684, P25683.

[0118] In a preferred embodiment, when calciseptin or toxin FS2 is used in the compositions of the present invention, the calciseptin or toxin FS2 has the sequence of SEQ ID NO: 26, SEQ ID NO: 27, or a derivative thereof.

[0119] The consensus sequence of Vasotab (SEQ ID NO: 28) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P84843, C1IBZ2, A0A0K8TPQ2, C8YJB4, C8YJB3.

[0120] In a preferred embodiment, when Vasotab is used in the compositions of the present invention, the Vasotab has the sequence of SEQ ID NO: 29, or a derivative thereof.

[0121] The consensus sequence of gulacontryphan M (SEQ ID NO: 30) has been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: U6C1Y3, A0A0K8TU78, A0A1P8NVU1, F5C3T9, A0A1P8NVS9, A0A1P8NVU0.

[0122] In a preferred embodiment, when glacontryphan M is used in the compositions of the present invention, it has the sequence of SEQ ID NO: 31, or a derivative thereof.

[0123] The consensus sequence of ω-ctenitoxin-Cs1a (SEQ ID NO: 33) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P81694, B6DCP3, B6DCP1, B6DCP4, B6DCN7.

[0124] In a preferred embodiment, when ω-ctenitoxin-Cs1a is used in the compositions of the present invention, the ω-ctenitoxin-Cs1a has the sequence of SEQ ID NO: 34, or a derivative thereof.

[0125] The consensus sequences of U9-ctenitoxin-Pn1a, U6-ctenitoxin-Pn1a, and κ-ctenitoxin-Pn1a (SEQ ID NO: 36) have been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: O76200, O76201, O76201, P0C2S6, P84000, P83895, P30288, P37045, P81793.

[0126] In a preferred embodiment, when U9-ctenitoxin-Pn1a, or U6-ctenitoxin-Pn1a, or κ-ctenitoxin-Pn1a is used in the compositions of the present invention, the U9-ctenitoxin-Pn1a, or U6-ctenitoxin-Pn1a, or κ-ctenitoxin-Pn1a has the sequence of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or a derivative thereof.

[0127] The consensus sequence of ω-ctenitoxin-Pr2a (SEQ ID NO: 40) has been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P81792, P83902, P0C2S7, P84011, P83901.

[0128] In a preferred embodiment, when ω-ctenitoxin-Pr2a is used in the compositions of the present invention, the ω-ctenitoxin-Pr2a has the sequence of SEQ ID NO: 41, or a derivative thereof.

[0129] The consensus sequence of toxin S4C8 and toxin C10S2C2 (SEQ ID NO: 42) has been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P22947, P01414, P25684, P25683.

[0130] In a preferred embodiment, when the toxin S4C8 or the toxin C10S2C2 is used in the compositions of the invention, the toxin S4C8 or the toxin C10S2C2 has the sequence of SEQ ID NO: 43, SEQ ID NO: 44, or a derivative thereof.

[0131] In an even more preferred embodiment, when the target receptor expressed by skeletal muscle cells is CaV1.1, the at least one postsynaptic peptide used in the compositions of the present invention is U7-ctenitoxin-Pn1b having the sequence of SEQ ID NO: 35, or calciceptin having the sequence of SEQ ID NO: 25 or SEQ ID NO: 26, or a derivative thereof.

[0132] Nav1.4 channels are found primarily in the sarcolemma of skeletal muscle fibers and in the T-tubule membrane. Depolarizing currents passing through the pores of these channels initiate action potentials in skeletal muscle, resulting in contraction.

[0133] Mu-conotoxins (μ-conotoxins) directly attenuate muscle action potentials by binding to Nav1.4 channels. Mu-conopeptides are isolated from the venom of marine snails in the genus Conus. The primary structure of μ-conopeptides consists of 15–30 amino acids folded with disulfide bridges. μ-conotoxins have a characteristic CC-Xm-C-Xn-C-Xp-CC framework, with six cysteines providing three possible disulfide bonds (i.e., CysI-CysIV, CysII-CysV, and CysIII-CysVI). The number of residues contained within the three loops (m, n, p) of μ-conotoxins provides the basis for dividing them into several structural subgroups (m can vary from 5–9 amino acid residues, n can vary from 3–4 amino acid residues, and p can vary from 3–5 amino acid residues).

[0134] In some embodiments, postsynaptic peptides capable of binding to the NaV1.4 channel and suitable for the compositions of the invention can be selected from the group comprising or consisting of μ-conotoxin GIIIb (SEQ ID NO:45 or SEQ ID NO:46), μ-conotoxin TIIIa (SEQ ID NO:45 or SEQ ID NO:48), μ-conotoxin PIIIa (SEQ ID NO:45 or SEQ ID NO:47), μ-conotoxin GvIIJ (SEQ ID NO:49 or SEQ ID NO:50), μ-O-conotoxin MfVIA (SEQ ID NO:51 or SEQ ID NO:52), μ-conotoxin CnIIIc (SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:95), μ-tomitoxin-Hme1a (SEQ ID NO:55 or SEQ ID NO:56), μ-tomitoxin-Hme1b (SEQ ID NO:55 or SEQ ID NO:57), μ-tomitoxin-Hme1c (SEQ ID NO:55 or SEQ ID NO:58), or derivatives thereof.

[0135] The consensus sequences of μ-conotoxin GIIIb, μ-conotoxin TIIIA, and μ-conotoxin PIIIa (SEQ ID NO: 45) have been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P01523, P01524, P05482, P0C349, P0DKQ9, P0DKQ9, X5IGY9, A0A0K8TUB1, P0CH16, P58925.

[0136] In a preferred embodiment, when μ-conotoxin GIIIb or μ-conotoxin TIIIA, or μ-conotoxin PIIIa is used in the composition of the present invention, the μ-conotoxin GIIIb or μ-conotoxin TIIIA, or μ-conotoxin PIIIa has the sequence of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 47, or a derivative thereof.

[0137] The consensus sequence of μ-conotoxin GvIIJ (SEQ ID NO: 49) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: X5IWS1, X5IA08, Q71KS8.

[0138] In a preferred embodiment, when mu-conotoxin GvIIJ is used in the compositions of the present invention, the mu-conotoxin GvIIJ has the sequence of SEQ ID NO: 50, or a derivative thereof.

[0139] The consensus sequence of mu-conotoxin CnIIIc (SEQ ID NO: 53) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: C1J5M5, C1J5M6, C1J5M7, I1SB07, P0CE77, Q86DU6, P60207, P0C1T9, P0C1U2, P0C195, P0C1U1, P0C1U0, P0CY74.

[0140] In a preferred embodiment, the postsynaptic peptide used in the compositions of the present invention is mu-conotoxin CnIIIc or a variant thereof.

[0141] In a more preferred embodiment, when mu-conotoxin CnIIIc is used in the compositions of the present invention, the mu-conotoxin CnIIIc has the sequence of SEQ ID NO: 54 or SEQ ID NO: 95, or a derivative thereof.

[0142] The consensus sequence of mu-O-conotoxin MfVIA (SEQ ID NO: 51) has been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P0DM15, A0A0K2S5L8, A0A0K2S5L9, A0A0K2S5N5, A0A0K2S6J2, A0A0K2S5L0, A0A0K2S5M2, A0A0K2S6H9, A0A0K2S5M9, A0A0K2S5M7, U6C1V9, P56708, U6C2E4, W4VSL3.

[0143] In a preferred embodiment, when mu-O-conotoxin MfVIA is used in the compositions of the invention, the mu-O-conotoxin MfVIA has the sequence of SEQ ID NO: 52, or a derivative thereof.

[0144] The consensus sequences of μ-tomitoxin Hme1a, μ-tomitoxin Hme1b, and μ-tomitoxin Hme1c (SEQ ID NO: 55) have been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P85505, P85506, COHJK5.

[0145] In a preferred embodiment, when μ-tomitoxin-Hme1a, μ-tomitoxin-Hme1b or μ-tomitoxin-Hme1c is used in the compositions of the present invention, μ-tomitoxin-Hme1a, μ-tomitoxin-Hme1b or μ-tomitoxin-Hme1c has the sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or a derivative thereof.

[0146] smooth muscle In another embodiment, when the postsynaptic cell is a smooth muscle cell, the at least one postsynaptic inhibitor of cholinergic neuronal transmission used in the compositions of the present invention binds to at least one receptor expressed by the smooth muscle cell, which receptor comprises or is selected from the group consisting of muscarinic acetylcholine receptors (mAChRs), more preferably mAChRs of the M3 subtype, ryanodine receptor type 2 (RYR2), voltage-gated L-type calcium channel CaV1.2, and voltage-gated sodium channel NaV1.5.

[0147] When two or more types of postsynaptic inhibitors of cholinergic neuronal transmission (PoNT) are used as components of the composition of the present invention, the receptors expressed by smooth muscle cells and targeted by these PoNTs may belong to the same receptor family or to multiple different receptor families.

[0148] Low molecular weight (smooth muscle) In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule, the synaptic inhibitor of neuronal transmission is selected according to the target postsynaptic receptor expressed by the smooth muscle cell, as shown in Table 3.

[0149] [Table 3(1)] [Table 3(2)]

[0150] Muscarinic acetylcholine receptors are an important subfamily of class A G protein-coupled receptors (GPCRs). There are five mAChR subtypes (M1–M5), which differ in their expression patterns, physiological functions, and G protein coupling. Three subtypes (M1, M3, and M5) transduce signals primarily through activation of G proteins of the Gq / 11 family, whereas M2 and M4 mAChRs transduce signals primarily through G proteins of the Gi / o family. M3 mAChRs are present in smooth muscle cells. Furthermore, muscarinic M3 receptors are thought to be the primary receptor subtype mediating parasympathetic contractile responses in smooth muscle tissue.

[0151] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to mAChR M3, the synaptic inhibitor of neuronal transmission comprises or is selected from the group consisting of atropine, scopolamine, hyoscyamine, ipratropium, tropicamide, flavoxate, oxybutynin, tiotropium, cyclopentolate, tolterodine, procyclidine, aclidinium, solifenacin, darifenacin, 4-DAMP, DAU-5884, J-104129, zamifenacin, or a combination thereof.

[0152] In a more preferred embodiment, when the target receptor expressed by smooth muscle cells is mAChR M3, the at least one postsynaptic small molecule used in the compositions of the present invention is darifenacin.

[0153] Ryanodine receptor isoform-2 (RyR2) is one of the three major calcium channels in smooth muscle that is important for excitation-contraction coupling. The contribution of RyR2 to calcium spark generation in smooth muscle is well described.

[0154] In a more preferred embodiment, when the target receptor expressed by smooth muscle cells is RyR2, the at least one postsynaptic small molecule used in the compositions of the present invention is JTV519.

[0155] The voltage-gated L-type calcium channel CaV1.2 is a calcium channel found in the membrane of smooth muscle cells. CaV1.2 is a protein complex that is integral to the cell membrane and acts to transport Ca into the cell in response to membrane depolarization. 2+ In smooth muscle cells, it binds to the ryanodine receptor RyR2 in the sarcoplasmic reticulum via mechanical coupling.

[0156] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to CaV1.2, the postsynaptic small molecule comprises or is selected from the group consisting of dihydropyridine, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, elgodipine, felodipine, flordipine, iganidipine, isradipine, lacidipine, lercanidipine, levumlodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, riodipine, verapamil, gallopamil, dimeditiapramine, diltiazem, or a combination thereof.

[0157] In a more preferred embodiment, when the target receptor expressed by smooth muscle cells is CaV1.2, the at least one postsynaptic small molecule used in the compositions of the present invention is verapamil.

[0158] Postsynaptic peptides (smooth muscle) In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic peptide, the synaptic inhibitor of neuronal transmission is selected according to the target postsynaptic receptor expressed by the smooth muscle cell, as shown in Table 4.

[0159] [Table 4]

[0160] The consensus sequence of muscarinic toxin alpha (SEQ ID NO: 59) is defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P80494, Q9PSN1, P81030, P86419, P80495, P85092, P25518, P17696, P82463, P60234, P18328, Q8QGR0, P81031.

[0161] A postsynaptic peptide capable of binding to mAChR M3 and suitable for the compositions of the invention is a muscarinic toxin (SEQ ID NO: 59 or SEQ ID NO: 60), or a derivative thereof.

[0162] Postsynaptic peptides capable of binding to RyR2 and suitable for the compositions of the invention may be selected from the group comprising or consisting of impellacarcin (SEQ ID NO: 15 or SEQ ID NO: 16), phospholipase A2 imperatoxin-1 (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 18), heteromutoxin (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 19), phospholipase A2 (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 20), phospholipase A2 (SEQ ID NO: 17 or SEQ ID NO: 21), insecticidal toxin LaIT1 (SEQ ID NO: 22 or SEQ ID NO: 23) and Φ-liotoxin LW1a (SEQ ID NO: 22 or SEQ ID NO: 24), or derivatives thereof.

[0163] In a preferred embodiment, when impellacalcin is used in the compositions of the present invention, the impellacalcin has the sequence of SEQ ID NO: 16, or a derivative thereof.

[0164] In a preferred embodiment, when phospholipase A2 imperatoxin-1 (large subunit), or heteromutoxin (large subunit), or phospholipase A2 (large subunit), or phospholipase A2 is used in the compositions of the present invention, the phospholipase A2 imperatoxin-1 (large subunit), or heteromutoxin (large subunit), or phospholipase A2 (large subunit), or phospholipase A2 has the sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or a derivative thereof.

[0165] In a preferred embodiment, when the insecticidal toxin LaIT1 or φ-liotoxin LW1a is used in the composition of the present invention, the insecticidal toxin LaIT1 or φ-liotoxin LW1a has the sequence of SEQ ID NO: 23, SEQ ID NO: 24, or a derivative thereof.

[0166] In an even more preferred embodiment, when the target postsynaptic receptor expressed by smooth muscle cells is RyR2, the at least one postsynaptic peptide used in the compositions of the present invention is the insecticidal toxin LaIT1 having the sequence of SEQ ID NO: 22, SEQ ID NO: 23, or a derivative thereof.

[0167] Postsynaptic peptides capable of binding to CaV1.2 and suitable for compositions of the invention include calciceptin (SEQ ID NO: 25 or SEQ ID NO: 26), toxin FS2 (SEQ ID NO: 25 or SEQ ID NO: 27), vasotab (SEQ ID NO: 28 or SEQ ID NO: 29), glacontryphan M (SEQ ID NO: 30 or SEQ ID NO: 31), ω-conotoxin-TxVII (SEQ ID NO: 32), ω-ctenitoxin-Cs1a (SEQ ID NO: 33 or SEQ ID NO: 34), U7-ctenitoxin-Pn1b (SEQ ID NO: 35), U9-ctenitoxin-Pn 1a (SEQ ID NO:36 or SEQ ID NO:37), U6-ctenitoxin-Pn1a (SEQ ID NO:36 or SEQ ID NO:38), κ-ctenitoxin-Pn1a (SEQ ID NO:36 or SEQ ID NO:39), ω-ctenitoxin-Pr2a (SEQ ID NO:40 or SEQ ID NO:41), toxin S4C8 (SEQ ID NO:42 or SEQ ID NO:43), toxin C10S2C2 (SEQ ID NO:42 or SEQ ID NO:44) and ω-terafotoxin-Cc1a (SEQ ID NO:61), or a derivative thereof.

[0168] In a preferred embodiment, when calciseptin or toxin FS2 is used in the compositions of the present invention, the calciseptin or toxin FS2 has the sequence of SEQ ID NO: 26, SEQ ID NO: 27, or a derivative thereof.

[0169] In a preferred embodiment, when Vasotab is used in the compositions of the present invention, the Vasotab has the sequence of SEQ ID NO: 29, or a derivative thereof.

[0170] In a preferred embodiment, when glacontryphan M is used in the compositions of the present invention, it has the sequence of SEQ ID NO: 31, or a derivative thereof.

[0171] In a preferred embodiment, when ω-ctenitoxin-Cs1a is used in the compositions of the present invention, the ω-ctenitoxin-Cs1a has the sequence of SEQ ID NO: 34, or a derivative thereof.

[0172] In a preferred embodiment, when U9-ctenitoxin-Pn1a, or U6-ctenitoxin-Pn1a, or κ-ctenitoxin-Pn1a is used in the compositions of the present invention, the U9-ctenitoxin-Pn1a, or U6-ctenitoxin-Pn1a, or κ-ctenitoxin-Pn1a has the sequence of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or a derivative thereof.

[0173] In a preferred embodiment, when ω-ctenitoxin-Pr2a is used in the compositions of the present invention, the ω-ctenitoxin-Pr2a has the sequence of SEQ ID NO: 41, or a derivative thereof.

[0174] In a preferred embodiment, when the toxin S4C8 or the toxin C10S2C2 is used in the compositions of the invention, the toxin S4C8 or the toxin C10S2C2 has the sequence of SEQ ID NO: 43, SEQ ID NO: 44, or a derivative thereof.

[0175] In an even more preferred embodiment, when the target postsynaptic receptor expressed by smooth muscle cells is CaV1.2, the at least one postsynaptic peptide used in the compositions of the present invention is U7-ctenitoxin-Pn1b having the sequence of SEQ ID NO: 35, or calciceptin having the sequence of SEQ ID NO: 25 or SEQ ID NO: 26, or a derivative thereof.

[0176] When greater specificity for the target postsynaptic receptor CaV1.2 is required, at least one postsynaptic peptide used in the compositions of the present invention is ω-terafotoxin-Cc1a (SEQ ID NO: 61), or a derivative thereof.

[0177] NaV1.5 (encoded by the SCN5A gene) is expressed in human smooth muscle cells and interstitial cells of Cajal (ICCs) in the small intestine and colon. Among other functions, ICCs act as pacemakers for the gastrointestinal tract, generating periodic depolarizations (slow waves) that are transmitted to smooth muscle, providing the electrical impulse for contraction. NaV channels are thought to be excitable by slow waves in human gastrointestinal smooth muscle. Furthermore, pharmacological blockade of NaV1.5 leads to constipation, suggesting that NaV1.5 is functionally important in the human gastrointestinal tract.

[0178] Postsynaptic peptides capable of binding to NaV1.5 and suitable for the compositions of the invention can be selected from the group comprising or consisting of β-mammalian toxin Css2 (SEQ ID NO:62 or SEQ ID NO:63), α-like toxin BmK-M1 (SEQ ID NO:64 or SEQ ID NO:65), μ-tomitoxin-Hme1a (SEQ ID NO:55 or SEQ ID NO:56), μ-tomitoxin-Hme1b (SEQ ID NO:55 or SEQ ID NO:57), μ-tomitoxin-Hme1c (SEQ ID NO:55 or SEQ ID NO:58), β / κ-terafotoxin-Cg1a (SEQ ID NO:66 or SEQ ID NO:67), κ-terafotoxin-Cg1a (SEQ ID NO:68 or SEQ ID NO:69), δ-terafotoxin-Cg1a 1 (SEQ ID NO:70 or SEQ ID NO:71), δ-terafotoxin-Cg1a 2 (SEQ ID NO:70 or SEQ ID NO:72), and δ-terafotoxin-Cg1a 3 (SEQ ID NO:70 or SEQ ID NO:73), or derivatives thereof.

[0179] The consensus sequence of mammalian toxin Css2 (SEQ ID NO: 62) has been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P08900, P01495, P56646, P60267, P59897, P18926, P59898, P60266, P0DL83, P0CH41, P45662, P45666, Q95WD2, P80076, Q7Z1K9, Q7Z1K8, Q9TWL0, C0HK69.

[0180] In a preferred embodiment, when β-mammalian toxin Css2 is used in the compositions of the present invention, the β-mammalian toxin Css2 has the sequence of SEQ ID NO: 63, or a derivative thereof.

[0181] The consensus sequence of the alpha-like toxin BmK-M1 (SEQ ID NO: 64) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: Q9GQV6, P59360, P58488, Q6IZE0, P59354, P01488, G4WFQ2, P01487, P17728, B8XGY6, Q9N682, P01483, P09981, B6A8S0, B6A8R9, P58328, P15227, P45698, Q9NJC8, P86405, P59896, E7D081, E7D082, P55902, D8UWD5, D8UWD4, P0DJH8, D8UWD6, P60257, O61705, Q9NJC5, P60255, P01489, P84646, B8XGX9, P83644, P84614.

[0182] In a preferred embodiment, when the alpha-like toxin BmK-M1 is used in the compositions of the present invention, the alpha-like toxin BmK-M1 has the sequence of SEQ ID NO: 65, or a derivative thereof.

[0183] The consensus sequence of β / κ-terafotoxin-Cg1a (SEQ ID NO: 66) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P62520, P84836, P84835, B1P1C8, B1P1D0, B1P1C9, P0CH52, P60980.

[0184] In a preferred embodiment, when β / κ-terafotoxin-Cg1a is used in the compositions of the present invention, the β / κ-terafotoxin-Cg1a has the sequence of SEQ ID NO: 67, or a derivative thereof.

[0185] The consensus sequence of κ-terafotoxin-Cg1a (SEQ ID NO: 68) was defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P0C247, B1P1E4, B1P1H1, B1P1A0.

[0186] In a preferred embodiment, when κ-terafotoxin-Cg1a is used in the compositions of the present invention, the κ-terafotoxin-Cg1a has the sequence of SEQ ID NO: 69, or a derivative thereof.

[0187] The consensus sequences of δ-terafotoxin-Cg1a 1, δ-terafotoxin-Cg1a 2, and δ-terafotoxin-Cg1a 3 (SEQ ID NO: 70) have been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: B1P1B7, B1P1B8, A0A482ZD06, B1P1B9.

[0188] In a preferred embodiment, when δ-terafotoxin-Cg1a 1, or δ-terafotoxin-Cg1a 2, or δ-terafotoxin-Cg1a 3 is used in the compositions of the present invention, the δ-terafotoxin-Cg1a 1, or δ-terafotoxin-Cg1a 2, or δ-terafotoxin-Cg1a 3 has the sequence of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or a derivative thereof.

[0189] In a preferred embodiment, when μ-tomitoxin-Hme1a, μ-tomitoxin-Hme1b, or μ-tomitoxin-Hme1c is used in the compositions of the present invention, the μ-tomitoxin-Hme1a, μ-tomitoxin-Hme1b, or μ-tomitoxin-Hme1c has the sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or a derivative thereof.

[0190] In an even more preferred embodiment, when the target postsynaptic receptor expressed by smooth muscle cells is Nav1.5, the at least one postsynaptic peptide used in the compositions of the present invention is κ-terafotoxin-Cg1a having the sequence 68 or SEQ ID NO: 69, or β-mammalian toxin Css2 having the sequence of SEQ ID NO: 62 or SEQ ID NO: 63, or a derivative thereof.

[0191] myocardium In another embodiment, when the postsynaptic cell is a cardiomyocyte, the at least one postsynaptic inhibitor of cholinergic neuronal transmission used in the compositions of the present invention binds to at least one receptor expressed by the cardiomyocyte, which receptor comprises or is selected from the group consisting of muscarinic acetylcholine receptors (mAChRs), more preferably M2 subtype mAChRs, ryanodine receptor type 2 (RYR2), voltage-gated L-type calcium channels CaV1.1 or CaV1.2, and voltage-gated sodium channels NaV1.5.

[0192] When two or more postsynaptic inhibitors of cholinergic neuronal transmission (PoNT) are used as components of the composition of the present invention, the receptors expressed by cardiomyocytes and targeted by these PoNTs may belong to the same receptor family or to several different receptor families.

[0193] Small molecules (myocardium) In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule, the synaptic inhibitor of neuronal transmission is selected according to the target postsynaptic receptor expressed by the cardiomyocyte, as shown in Table 5.

[0194] [Table 5(1)] [Table 5(2)]

[0195] M2 muscarinic acetylcholine receptors are present in cardiac myocytes and regulate cardiac rhythm, particularly through modulation of inward potassium currents.

[0196] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to mAChR M2, the at least one synaptic inhibitor of neuronal transmission is selected from the group comprising or consisting of atropine, scopolamine, hyoscyamine, ipratropium, tropicamide, flavoxate, oxybutynin, tiotropium, cyclopentolate, tolterodine, procyclidine, aclidinium, AFDX 116, AFDX-384, methoctramine, otenzepad, tripitramine, or a combination thereof.

[0197] In a more preferred embodiment, when the target postsynaptic receptor expressed by cardiomyocytes is mAChR M2, the at least one postsynaptic small molecule used in the compositions of the invention is tolterodine.

[0198] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to RyR2, the at least one postsynaptic small molecule used in the compositions of the invention is JTV519.

[0199] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to CaV1.1 or CaV1.2, the synaptic inhibitor of neuronal transmission comprises or is selected from the group consisting of dihydropyridine, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, elgodipine, felodipine, flordipine, iganidipine, isradipine, lacidipine, lercanidipine, levumlodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, riodipine, verapamil, gallopamil, dimeditiapramine, diltiazem, or a combination thereof.

[0200] In a more preferred embodiment, when the target receptor expressed by smooth muscle cells is CaV1.1 or CaV1.2, the at least one postsynaptic small molecule used in the compositions of the present invention is verapamil.

[0201] Postsynaptic peptides (cardiac muscle) In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic peptide, the synaptic inhibitor of neuronal transmission is selected according to the target postsynaptic receptor expressed by the cardiomyocyte, as shown in Table 6.

[0202] [Table 6]

[0203] A postsynaptic peptide capable of binding to mAChR M2 and suitable for the compositions of the invention is muscarinic toxin alpha having the sequence of SEQ ID NO: 59, SEQ ID NO: 60, or a derivative thereof.

[0204] Suitable postsynaptic peptides capable of binding to CaV1.1 or CaV1.1 that are suitable for the compositions of the invention include calciceptin (SEQ ID NO: 25 or SEQ ID NO: 26), toxin FS2 (SEQ ID NO: 25 or SEQ ID NO: 27), vasotab (SEQ ID NO: 28 or SEQ ID NO: 29), glacontriphan M (SEQ ID NO: 30 or SEQ ID NO: 31), ω-conotoxin-TxVII (SEQ ID NO: 32), ω-ctenitoxin-Cs1a (SEQ ID NO: 33 or SEQ ID NO: 34), U7-ctenitoxin-Pn1b (SEQ ID NO: 35), U9-ctenitoxin-TxVII (SEQ ID NO: 36), U1-ctenitoxin-Pn1b (SEQ ID NO: 37), U2-ctenitoxin-Pn1c (SEQ ID NO: 38), U3-ctenitoxin-Pn1d (SEQ ID NO: 39), U4-ctenitoxin-Pn1e (SEQ ID NO: 40), U5-ctenitoxin-Pn1f (SEQ ID NO: 41), U6-ctenitoxin-Pn1g (SEQ ID NO: 42), U7-ctenitoxin-Pn1i (SEQ ID NO: 43), U8-ctenitoxin-Pn1i (SEQ ID NO: 44), U9-ctenitoxin-Pn1i (SEQ ID NO: 45), U1-ctenitoxin-Pn1i (SEQ ID NO: 46), U1-ctenitoxin-Pn1i (SEQ ID NO: 47), U2-ctenitoxin-Pn1i (SEQ ID NO: 48), U3-ctenitox The toxin may be selected from the group comprising or consisting of U6-ctenitoxin-Pn1a (SEQ ID NO:36 or SEQ ID NO:37), U6-ctenitoxin-Pn1a (SEQ ID NO:36 or SEQ ID NO:38), κ-ctenitoxin-Pn1a (SEQ ID NO:36 or SEQ ID NO:39), ω-ctenitoxin-Pr2a (SEQ ID NO:40 or SEQ ID NO:41), toxin S4C8 (SEQ ID NO:42 or SEQ ID NO:43) and toxin C10S2C2 (SEQ ID NO:42 or SEQ ID NO:44), ω-terafotoxin-Cc1a (SEQ ID NO:61), or a derivative thereof.

[0205] In a preferred embodiment, when calciseptin or toxin FS2 is used in the compositions of the present invention, the calciseptin or toxin FS2 has the sequence of SEQ ID NO: 25, SEQ ID NO: 26, or a derivative thereof.

[0206] In a preferred embodiment, when Vasotab is used in the compositions of the present invention, the Vasotab has the sequence of SEQ ID NO: 28, SEQ ID NO: 29, or a derivative thereof.

[0207] In a preferred embodiment, when glacontryphan M is used in the compositions of the present invention, it has the sequence of SEQ ID NO: 30, SEQ ID NO: 31, or a derivative thereof.

[0208] In a preferred embodiment, when ω-conotoxin-TxVII is used in the compositions of the present invention, the ω-conotoxin-TxVII has the sequence of SEQ ID NO: 32, or a derivative thereof.

[0209] In a preferred embodiment, when ω-ctenitoxin-Cs1a is used in the compositions of the present invention, the ω-ctenitoxin-Cs1a has the sequence of SEQ ID NO: 33, SEQ ID NO: 34, or a derivative thereof.

[0210] In a preferred embodiment, when U9-ctenitoxin-Pn1a, U6-ctenitoxin-Pn1a, or κ-ctenitoxin-Pn1a is used in the compositions of the present invention, the U9-ctenitoxin-Pn1a, U6-ctenitoxin-Pn1a, or κ-ctenitoxin-Pn1a has the sequence of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or a derivative thereof.

[0211] In a preferred embodiment, when ω-ctenitoxin-Pr2a is used in the compositions of the present invention, the ω-ctenitoxin-Pr2a has the sequence of SEQ ID NO: 40, SEQ ID NO: 41, or a derivative thereof.

[0212] In a preferred embodiment, when the toxin S4C8 or the toxin C10S2C2 is used in the compositions of the invention, the toxin S4C8 or the toxin C10S2C2 has the sequence of SEQ ID NO: 43, SEQ ID NO: 44, or a derivative thereof.

[0213] In an even more preferred embodiment, when the target postsynaptic receptor expressed by cardiomyocytes is CaV1.1 or CaV1.2, the at least one postsynaptic peptide used in the compositions of the present invention is U7-ctenitoxin-Pn1b having SEQ ID NO: 35, or calciceptin having SEQ ID NO: 25 or SEQ ID NO: 26, or a derivative thereof.

[0214] When greater specificity for the target postsynaptic receptor CaV1.2 is required, at least one postsynaptic peptide used in the compositions of the present invention is ω-terafotoxin-Cc1a having SEQ ID NO: 61, or a derivative thereof.

[0215] Postsynaptic peptides capable of binding to RyR2 and suitable for the compositions of the invention may be selected from the group comprising or consisting of impellacarcin (SEQ ID NO: 15 or SEQ ID NO: 16), phospholipase A2 imperatoxin-1 (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 18), heteromutoxin (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 19), phospholipase A2 (large subunit) (SEQ ID NO: 17 or SEQ ID NO: 20), phospholipase A2 (SEQ ID NO: 17 or SEQ ID NO: 21), insecticidal toxin LaIT1 (SEQ ID NO: 22 or SEQ ID NO: 23) and Φ-liotoxin LW1a (SEQ ID NO: 22 or SEQ ID NO: 24), or derivatives thereof.

[0216] In a preferred embodiment, when impellacalcin is used in the compositions of the present invention, the impellacalcin has the sequence of SEQ ID NO: 16, or a derivative thereof.

[0217] In a preferred embodiment, when phospholipase A2 imperatoxin-1 (large subunit), or heteromutoxin (large subunit), or phospholipase A2 (large subunit), or phospholipase A2 is used in the compositions of the present invention, the phospholipase A2 imperatoxin-1 (large subunit), or heteromutoxin (large subunit), or phospholipase A2 (large subunit), or phospholipase A2 has the sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or a derivative thereof.

[0218] In a preferred embodiment, when the insecticidal toxin LaIT1 or φ-liotoxin LW1a is used in the composition of the present invention, the insecticidal toxin LaIT1 or φ-liotoxin LW1a has the sequence of SEQ ID NO: 23, SEQ ID NO: 24, or a derivative thereof.

[0219] In an even more preferred embodiment, when the target postsynaptic receptor expressed by cardiomyocytes is RyR2, the at least one postsynaptic peptide used in the compositions of the present invention is the insecticidal toxin LaIT1 having the sequence of SEQ ID NO: 22, SEQ ID NO: 23, or a derivative thereof.

[0220] Postsynaptic peptides capable of binding to NaV1.5 and suitable for the compositions of the invention can be selected from the group comprising or consisting of β-mammalian toxin Css2 (SEQ ID NO:62 or SEQ ID NO:63), α-like toxin BmK-M1 (SEQ ID NO:64 or SEQ ID NO:65), μ-tomitoxin-Hme1a (SEQ ID NO:55 or SEQ ID NO:56), μ-tomitoxin-Hme1b (SEQ ID NO:55 or SEQ ID NO:57), μ-tomitoxin-Hme1c (SEQ ID NO:55 or SEQ ID NO:58), β / κ-terafotoxin-Cg1a (SEQ ID NO:66 or SEQ ID NO:67), κ-terafotoxin-Cg1a (SEQ ID NO:68 or SEQ ID NO:69), δ-terafotoxin-Cg1a 1 (SEQ ID NO:70 or SEQ ID NO:71), δ-terafotoxin-Cg1a 2 (SEQ ID NO:70 or SEQ ID NO:72), and δ-terafotoxin-Cg1a 3 (SEQ ID NO:70 or SEQ ID NO:73), or derivatives thereof.

[0221] In a preferred embodiment, when β-mammalian toxin Css2 is used in the compositions of the present invention, the β-mammalian toxin Css2 has the sequence of SEQ ID NO: 63, or a derivative thereof.

[0222] In a preferred embodiment, when the alpha-like toxin BmK-M1 is used in the composition of the present invention, the alpha-like toxin BmK-M1 has the sequence of SEQ ID NO: 65, or a derivative thereof.

[0223] In a preferred embodiment, when μ-tomitoxin-Hme1a, μ-tomitoxin-Hme1b, or μ-tomitoxin-Hme1c is used in the compositions of the present invention, the μ-tomitoxin-Hme1a, μ-tomitoxin-Hme1b, or μ-tomitoxin-Hme1c has the sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or a derivative thereof.

[0224] In a preferred embodiment, when β / κ-terafotoxin-Cg1a is used in the compositions of the present invention, the β / κ-terafotoxin-Cg1a has the sequence of SEQ ID NO: 67, or a derivative thereof.

[0225] In a preferred embodiment, when κ-terafotoxin-Cg1a is used in the compositions of the present invention, the κ-terafotoxin-Cg1a has the sequence of SEQ ID NO: 69, or a derivative thereof.

[0226] In a preferred embodiment, when δ-terafotoxin-Cg1a 1, or δ-terafotoxin-Cg1a 2, or δ-terafotoxin-Cg1a 3 is used in the compositions of the present invention, the δ-terafotoxin-Cg1a 1, or δ-terafotoxin-Cg1a 2, or δ-terafotoxin-Cg1a 3 has the sequence of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or a derivative thereof.

[0227] In a more preferred embodiment, when the target postsynaptic receptor expressed by cardiomyocytes is Nav1.5, the at least one postsynaptic peptide used in the compositions of the present invention is κ-terafotoxin-Cg1a having SEQ ID NO: 68 or SEQ ID NO: 69, or β-mammalian toxin Css2 having SEQ ID NO: 62 or SEQ ID NO: 63, or a derivative thereof.

[0228] secretory gland cells In another embodiment, when the postsynaptic cell is a secretory gland cell, the at least one postsynaptic inhibitor of cholinergic neuronal transmission used in the compositions of the present invention binds to at least one receptor expressed by the secretory gland cell, which receptor comprises or is selected from the group consisting of muscarinic acetylcholine receptors (mAChRs), more preferably mAChRs of the M1 or M3 subtype, alpha-1 adrenergic receptors, and beta-1 adrenergic receptors.

[0229] When two or more postsynaptic inhibitors of cholinergic neuronal transmission (PoNTs) are used as components of the composition of the present invention, the receptors expressed by the secretory cells and targeted by these PoNTs may belong to the same receptor family or to several different receptor families.

[0230] Low molecules (secretory glands) In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule, the synaptic inhibitor of neuronal transmission is selected according to the target postsynaptic receptor expressed by the secretory gland cell, as shown in Table 7.

[0231] [Table 7]

[0232] Several muscarinic receptor subtypes are involved in saliva secretion, including serous cells, which express a single population of M3 receptors, and mucous acinar cells, which express both M1 and M3 receptors.

[0233] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to mAChR M1 or M3, the synaptic inhibitor of neuronal transmission is selected from the group including or consisting of atropine, scopolamine, hyoscyamine, ipratropium, tropicamide, flavoxate, oxybutynin, tiotropium, cyclopentolate, tolterodine, procyclidine, aclidinium, or a combination thereof.

[0234] In a more preferred embodiment, when the target postsynaptic receptor expressed by the secretory cell is mAChR M1 or M3, the at least one postsynaptic small molecule used in the compositions of the present invention is oxybutynin.

[0235] Higher specificity for M1 or M3 mAchRs may be required.

[0236] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to mAChR M1, the synaptic inhibitor of neuronal transmission is selected from the group including or consisting of pirenzepine, telenzepine, benzhexol, or a combination thereof.

[0237] In a more preferred embodiment, when the target postsynaptic receptor expressed by the secretory gland cell is mAChR M1, the at least one postsynaptic small molecule used in the compositions of the present invention is pirenzepine, benzhexol, or a combination thereof.

[0238] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to mAChR M3, the synaptic inhibitor of neuronal transmission is selected from the group including or consisting of solifenacin, darifenacin, 4-DAMP, DAU-5884, J-104129, zamifenacin, or a combination thereof.

[0239] In a more preferred embodiment, when the target postsynaptic receptor expressed by the secretory cell is mAChR M3, the at least one postsynaptic small molecule used in the compositions of the present invention is darifenacin.

[0240] Adrenergic receptors (ARs) are a type of G protein-coupled receptor that bind to adenylyl cyclase. There are four general types of ARs (α1, α2, β1, and β2), which are found in different effector tissues. In salivary glands, α1 adrenergic receptors regulate the intracytoplasmic calcium concentration ([Ca2 + ]i) and stimulates saliva secretion. The α1-adrenergic pathway is also activated in the lacrimal gland.

[0241] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to alpha-adrenergic receptors, the synaptic inhibitor of neuronal transmission is selected from the group including or consisting of alfuzosin, doxazosin, indoramin, moxisylyte, prazosin, silodosin, tamsulosin, terazosin, or a combination thereof.

[0242] In a more preferred embodiment, when the target postsynaptic receptor expressed by the secretory gland cell is an α1-adrenergic receptor, the at least one postsynaptic small molecule used in the compositions of the present invention is silodosin.

[0243] In salivary glands, β1-adrenergic receptors are responsible for viscous secretions.

[0244] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic small molecule that binds to β1-adrenergic receptors, the synaptic inhibitor of neuronal transmission is selected from the group including or consisting of acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, nebivolol, vortioxetine, or a combination thereof.

[0245] In a more preferred embodiment, when the target postsynaptic receptor expressed by the secretory gland cell is the β1-adrenergic receptor, the at least one postsynaptic small molecule used in the compositions of the present invention is bisoprolol.

[0246] Postsynaptic peptides (secretory glands) In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic peptide, the synaptic inhibitor of neuronal transmission is selected according to the target postsynaptic receptor expressed by the secretory gland cell, as shown in Table 8.

[0247] [Table 8]

[0248] Postsynaptic peptides capable of binding to mAChr M1 suitable for the compositions of the invention can be selected from the group comprising or consisting of muscarinic toxin 1 (SEQ ID NO:59 or SEQ ID NO:74), muscarinic toxin 2 (SEQ ID NO:59 or SEQ ID NO:75), muscarinic toxin 3 (SEQ ID NO:59 or SEQ ID NO:76), muscarinic toxin 7 (SEQ ID NO:59 or SEQ ID NO:77), and muscarinic toxin alpha (SEQ ID NO:59 or SEQ ID NO:60), or derivatives thereof.

[0249] The consensus sequences of muscarinic toxin alpha, muscarinic toxin 1, muscarinic toxin 2, muscarinic toxin 3, and muscarinic toxin 7 (SEQ ID NO: 59) have been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P80494, Q9PSN1, P81030, P86419, P80495, P85092, P25518, P17696, P82463, P60234, P18328, Q8QGR0, P81031.

[0250] In a more preferred embodiment, when the postsynaptic receptor on the surface of the secretory gland cell is mAChr M1, the at least one postsynaptic peptide used in the compositions of the invention is muscarinic toxin 7 having the sequence of SEQ ID NO: 59, SEQ ID NO: 77, or a derivative thereof.

[0251] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic peptide that binds to mAChr M3, the at least one postsynaptic peptide used in the compositions of the invention is muscarinic toxin alpha having the sequence of SEQ ID NO: 59, SEQ ID NO: 60, or a derivative thereof.

[0252] Postsynaptic peptides capable of binding to alpha1-adrenergic receptors suitable for the compositions of the present invention can be selected from the group comprising or consisting of muscarinic toxin 1 (SEQ ID NO: 59 or SEQ ID NO: 74), muscarinic toxin 3 (SEQ ID NO: 59 or SEQ ID NO: 76), muscarinic toxin alpha (SEQ ID NO: 59 or SEQ ID NO: 60), adrenergic toxin ρ-elapitoxin-Dp1a (SEQ ID NO: 78), toxin AdTx1 (SEQ ID NO: 79), or derivatives thereof.

[0253] The consensus sequences of muscarinic toxin alpha, muscarinic toxin 1, muscarinic toxin 3, adrenergic toxin p-elapitoxin-Dp1a and toxin AdTx1 (SEQ ID NO: 59) have been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: P80494, Q9PSN1, P81030, P86419, P80495, P85092, P25518, P17696, P82463, P60234, P18328, Q8QGR0, P81031.

[0254] In a more preferred embodiment, when the postsynaptic receptor on the surface of the secretory gland cell is an α1-adrenergic receptor, the at least one postsynaptic peptide used in the composition of the present invention is the adrenergic toxin ρ-elapitoxin-Dp1a having the sequence of SEQ ID NO: 59 or SEQ ID NO: 78, or the toxin AdTx1 having the sequence of SEQ ID NO: 59 or SEQ ID NO: 79, or a derivative thereof.

[0255] Post-synaptic peptides capable of binding to β1-adrenergic receptors suitable for the compositions of the present invention may be selected from the group comprising or consisting of β-cardiotoxin (SEQ ID NO: 80 or SEQ ID NO: 81).

[0256] The consensus sequence of β-cardiotoxin (SEQ ID NO: 80) has been defined based on the sequences of proteins with the following accession numbers in the Uniprot database: Q69CK0, Q53B46, Q2VBN8, Q2VBN5, Q2VBN7, Q2VBN4.

[0257] In this embodiment, when the at least one synaptic inhibitor of neuronal transmission is a postsynaptic peptide that binds to β1-adrenergic receptors, the at least one postsynaptic peptide used in the compositions of the invention is β-cardiotoxin having the sequence of SEQ ID NO: 80, SEQ ID NO: 81, or a derivative thereof.

[0258] Multispecific postsynaptic inhibitors In other embodiments, postsynaptic inhibitors of neuronal transmission can be selected from small molecules and peptides that exhibit the ability to inhibit multiple molecular targets. Such "multispecific postsynaptic inhibitors" have activity against at least one of the aforementioned molecular targets (i.e., nAChR ((α1)2β1δε or (α1)2β1δγ), mAChR M1 or mAChR M2 or mAChR M3, α1-adrenergic receptor, β1-adrenergic receptor, RYR1 or RYR2, CaV1.1 or CaV1.2, NaV1.4 or NaV1.5). It is noteworthy that multispecificity can be advantageous in the present invention. For example, small molecules that primarily inhibit NaV can be used to reduce pain associated with a procedure or condition (since NaV1.7 is prominently involved in nociception).

[0259] In some embodiments, the multispecific postsynaptic inhibitor can be selected from the small molecules listed in Table 9.

[0260] [Table 9(1)] [Table 9(2)]

[0261] In a more preferred embodiment, the at least one multispecific postsynaptic small molecule used in the compositions of the present invention is lidocaine.

[0262] In other embodiments, the multispecific postsynaptic inhibitor can be selected from the peptides listed in Table 10, or derivatives thereof.

[0263] [Table 10]

[0264] In a more preferred embodiment, the at least one multispecific postsynaptic peptide used in the compositions of the present invention is β / ω-terafotoxin-Tp2a having the sequence of SEQ ID NO: 91, or a derivative thereof.

[0265] The present invention further provides a cosmetic composition comprising the composition of the present invention, which comprises at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin, and which may further comprise a cosmetically acceptable excipient or diluent.

[0266] The present invention further provides a pharmaceutical composition comprising a composition of the present invention comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin, which may further comprise a pharmaceutically acceptable excipient, diluent, carrier, salt and / or additive.

[0267] Embodiments and Other Aspects of the Invention BoNTs are reported to have direct effects that include inhibiting or blocking cholinergic neuromuscular or cholinergic autonomic innervation of secretory glands and smooth muscle.

[0268] The compositions of the present invention allow for experiments to modulate the pharmacodynamic profile of BoNT.

[0269] Rapid-onset PoNT Fast onset may be of particular interest for rapid relief of pain associated with muscle contractions.

[0270] Surprisingly, the inventors have found that the "early effect" of the muscle relaxant effect of the composition of the present invention is stronger than the "early effect" of each compound (i.e., BoNT and PoNT) administered independently. Thus, the composition of the present invention has a synergistic effect. "Early effect" refers to a muscle relaxant effect that occurs before the effect of BoNT, i.e., within the first 24 hours after BoNT administration.

[0271] In a preferred embodiment, the postsynaptic inhibitors of neuronal transmission (PoNT) used in the compositions of the present invention are fast-acting (fast-onset) postsynaptic peptides and / or fast-acting postsynaptic small molecules. "Fast-acting" refers to a PoNT that produces an effect more rapidly than the effect produced by a botulinum neurotoxin, e.g., BoNT / A, BoNT / B, or BoNT / E. For example, the effect of a fast-acting PoNT can be visually noticeable within 12 hours, preferably within 6 hours, more preferably within 2 hours, and most preferably within 1 hour.

[0272] In another preferred embodiment, the postsynaptic inhibitor of neuronal transmission (PoNT) used in the compositions of the present invention is a very fast-acting postsynaptic peptide or small molecule, where "very fast-acting" refers to a PoNT that provides an effect in less than 45 minutes, preferably less than 30 minutes, more preferably less than 20 minutes, and most preferably less than 10 minutes.

[0273] PoNT, which extends the duration of BoNT's action Even more surprisingly, the present inventors have discovered that the addition of at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, to a botulinum neurotoxin composition extends the duration of action of the botulinum neurotoxin. For example, the normal duration of action of BoNT / A after intramuscular injection is typically about 3 to 4 months in humans. The "extended duration of action" of a botulinum neurotoxin means that the action of the botulinum toxin lasts longer than, for example, the action of botulinum neurotoxin type A. The onset of action of the compositions of the present invention is delayed, for example, by 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or more, compared to the onset of action of a composition containing only BoNT.

[0274] This particular advantage of the present invention can be of particular interest for reducing the number of injections required to achieve a desired effect. Another advantage is that by increasing the time between two injections, the likelihood of anti-BoNT antibodies being produced by the immune system of an individual receiving a composition of the present invention is also reduced.

[0275] PoNT for increased strength Surprisingly, the inventors have discovered that administering at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, together with a botulinum neurotoxin composition enhances the potency of the action of the botulinum neurotoxin, where "potency of action" refers to the maximum observable muscle relaxant effect for a particular dose of BoNT.

[0276] In a preferred embodiment, the postsynaptic inhibitors of neuronal transmission (PoNT) used in the compositions of the present invention exhibit a synergistic effect that is at least 10%, preferably at least 25%, and more preferably at least 50% greater than the effect of the individual compounds of the composition administered independently.

[0277] By enhancing the potency of BoNT's action, the compositions of the present invention may improve cosmetic results and relief of disorders and painful conditions.

[0278] Rapid-onset BoNT, which accelerates BoNT onset, increases the potency of BoNT action, and increases the duration of action For some fast-acting or very fast-acting BoNTs, i.e., postsynaptic peptides or small postsynaptic molecules, it is possible to modulate the effect of the botulinum neurotoxin composition by accelerating the onset of action, extending the duration of action, and increasing the intensity of action.

[0279] Triple combination for rapid and sustained onset of action Depending on the intended use, if the duration of action of the PoNT ends before the maximum effect of the botulinum neurotoxin is achieved, the composition of the present invention may further comprise a second PoNT with an intermediate onset of action. "Intermediate onset of action" refers to a PoNT that provides an effect more slowly than a fast-acting PoNT. The advantage is that the rapid, sustained, and long-term maximum effect of the composition of the present invention is obtained without a significant decrease in these effects over time.

[0280] In a preferred embodiment, the composition of the present invention comprises at least one fast-acting PoNT, at least one intermediate-acting PoNT, and a botulinum neurotoxin.

[0281] Simultaneous administration In all embodiments, at least one PoNT and a botulinum neurotoxin are administered in combination. The terms "in combination," "in conjunction with," "co-delivery," and "administered together" are equivalent and may be used interchangeably. In the context of administering two or more components to a subject, the simultaneous administration of two or more components, for example, at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin composition, refers to the simultaneous administration of two or more components.

[0282] Co-administration of PoNT and BoNT is defined as the administration of at least one PoNT and one BoNT to the same anatomical site using similar or different routes of administration within a short time period, such as 5, 10, 15, 20, 25, or 30 minutes, or within a longer time period, such as 60, 120, 240, 360, or 720 minutes, regardless of whether the PoNT and BoNT are combined in the same composition or the same container.

[0283] The use of the term "in combination" does not restrict the order in which PoNT and BoNT are administered to a subject.

[0284] simultaneous injection All of the above-mentioned effects of using the compositions of the present invention are obtained when at least one PoNT, ie, a postsynaptic peptide or small post-synaptic molecule, and a botulinum neurotoxin are injected simultaneously.

[0285] Non-simultaneous injection In an alternative embodiment, at least one postsynaptic inhibitor of cholinergic neuronal transmission can be injected first, followed by a second injection with botulinum neurotoxin. In this embodiment, the injection of botulinum neurotoxin must occur before the end of the duration of action of the at least one postsynaptic inhibitor of cholinergic neuronal transmission. In other words, non-simultaneous injection of PoNT and botulinum neurotoxin may be performed, provided that the botulinum neurotoxin is administered before the decay of PoNT activity occurs, and optionally after the onset of PoNT activity.

[0286] In another alternative embodiment, the botulinum neurotoxin can be injected first, followed by a second injection with at least one postsynaptic inhibitor of cholinergic neuronal transmission, in which the at least one postsynaptic inhibitor of neuronal transmission must be injected before the effect of the botulinum neurotoxin reaches a plateau.

[0287] Topical administration Compared with 150KDa of BoNT, the PoNT used in the composition of the present invention has a low molecular weight.In the context of cosmetic treatment, it may be interesting to use different types of application methods for each component of the composition of the present invention.For example, at least one PoNT can be delivered by topical application.

[0288] In another embodiment, at least one postsynaptic inhibitor of cholinergic neuronal transmission can be applied topically to the skin near the target tissue of interest, and the botulinum neurotoxin is injected into the target tissue of interest. In this embodiment, the injection of the botulinum neurotoxin must occur before the end of the duration of action of the at least one postsynaptic inhibitor of cholinergic neuronal transmission.

[0289] Injection Schedule The injection schedule must also be compatible with the pharmacodynamic profile of PoNT so that it can interact with the botulinum neurotoxin at the synaptic level in the target tissue.

[0290] A second object of the present invention relates to a method for enhancing the effect of a botulinum neurotoxin composition, which method comprises adding at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, to the botulinum neurotoxin composition.

[0291] "Enhancing the effect of a botulinum neurotoxin composition" means accelerating the onset of action of the botulinum neurotoxin composition, and / or extending the duration of action, and / or increasing the intensity of the action.

[0292] Accordingly, the present invention relates to a method for accelerating the onset of action and / or prolonging the duration of action and / or increasing the potency of action of a botulinum neurotoxin composition, which method comprises adding at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, to the botulinum neurotoxin composition.

[0293] Onset of action "Accelerating the onset" of action of a botulinum neurotoxin composition means obtaining muscle relaxation more quickly than would be obtained with BoNT alone.

[0294] Preferably, the onset of action of a botulinum neurotoxin composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, occurs 50%, preferably 75%, and more preferably 90% faster than the duration of action of a botulinum neurotoxin composition comprising only a BoNT (i.e., no postsynaptic inhibitor of cholinergic neuronal transmission).

[0295] Since the same botulinum neurotoxin is used in both compositions, the botulinum neurotoxin composition containing BoNT alone serves as a relevant control for comparison.

[0296] Duration "Extending the duration" of action of a botulinum neurotoxin composition means that at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, delays recovery from BoNT-induced muscle relaxation. In other words, the duration of muscle relaxation following intramuscular injection of a composition of the invention may be considered "long recovery."

[0297] Preferably, the duration of action of a botulinum neurotoxin composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, is extended by 10%, 25%, 50%, 100% or more compared to the duration of action of a botulinum neurotoxin composition comprising BoNT alone (i.e., without a postsynaptic inhibitor of cholinergic neuronal transmission).

[0298] Since the same botulinum neurotoxin is used in both compositions, the botulinum neurotoxin composition containing BoNT alone serves as a relevant control for comparison.

[0299] strength In some embodiments, a third effect of the at least one postsynaptic inhibitor of cholinergic neuronal transmission may be to increase the maximum magnitude of action of the botulinum toxin, preferably by at least 2%, preferably at least 5%, and more preferably at least 10% compared to the maximum magnitude of action of a composition containing only botulinum neurotoxin (i.e., no postsynaptic inhibitor of cholinergic neuronal transmission).

[0300] Since the same botulinum neurotoxin is used in both compositions, the botulinum neurotoxin composition containing BoNT alone serves as a relevant control for comparison.

[0301] This third effect may be particularly interesting, for example, to achieve better relief in patients with severe dystonia. Another advantage is the fact that the compositions of the present invention allow for satisfactory effects with lower doses of BoNT, thus reducing the risk of toxicity associated with BoNT overdose (especially in Parkinson's patients who require multiple injections in various parts of the body).

[0302] Onset, intensity and duration of action In some embodiments, at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, has a triple effect on the botulinum neurotoxin composition: accelerating the onset of action of the botulinum neurotoxin composition, prolonging the duration of action, and increasing the potency of the action.

[0303] The compositions of the present invention may further comprise other ingredients suitable for cosmetic or therapeutic use.

[0304] formulation A third object of the present invention is a cosmetic or pharmaceutical composition comprising the composition of the present invention and one or more dermatologically or pharmaceutically acceptable carriers, such as, but not limited to, albumin, preferably human serum albumin or recombinant human albumin, non-reducing di- or trisaccharides, non-ionic surfactants, and any other salts and / or additives suitable for the intended use.

[0305] The compositions of the invention are preferably in a form that allows for subcutaneous, intramuscular, implant and / or topical administration, with intramuscular and subcutaneous administration being preferred.

[0306] Compositions of the present invention suitable for injection (infusion) into a subject or patient (i.e., a human or other mammal in need of a particular treatment) may be in the form of a solution, suspension, emulsion, or dry powder that is dissolved or suspended in a suitable vehicle prior to use. The term "in need" is meant to include both pharmaceutical or health-related needs (e.g., treating conditions related to dystonia or spasticity) and cosmetic and subjective needs (e.g., altering or improving appearance, such as treating facial wrinkles).

[0307] When delivering postsynaptic peptides locally, they may be formulated into creams, foams, gels, lotions, ointments (e.g., for topical application), or for subcutaneous injection. Local delivery means may also include transdermal delivery (e.g., via adhesive patches, iontophoresis, or ultrasound devices).

[0308] The compositions of the present invention are suitable for local delivery. "Local delivery" means that the site of action is at or near the site of use (application) of the composition. As a non-limiting example, a composition comprising at least one PoNT and a botulinum neurotoxin injected into a muscle to be relaxed will be delivered locally to that muscle by intramuscular injection.

[0309] PoNT and BoNT mixture In a preferred embodiment, the composition is prepared by mixing a botulinum toxin (with or without associated non-toxin proteins) with at least one postsynaptic inhibitor of cholinergic neuronal transmission, and typically one or more additional pharmaceutically acceptable carriers or excipients. In its simplest form, the composition may contain an aqueous, pharmaceutically acceptable diluent, such as buffered saline. However, the composition may also contain other ingredients typically found in injectable pharmaceutical or cosmetic compositions, including dermatologically or pharmaceutically acceptable carriers, vehicles, or media that are compatible with the tissues to which it is applied. As used herein, the term "dermatologically or pharmaceutically acceptable" means that the composition or its components so described are suitable for use in contact with these tissues or for use by patients in general, without undue toxicity, incompatibility, instability, allergic reaction, etc. If desired, the compositions of the present invention may contain any ingredients conventionally used in the fields of interest, particularly cosmetics and dermatology.

[0310] Administration to skeletal muscle The pharmaceutical compositions of the present invention are suitable for injection into or near one or more of the following muscles: for example, corrugator supercilii, procerus, occipitofrontalis, nasalis, orbicularis oris, depressor anguli oris, platysma, sternohyoid, serratus anterior, rectus abdominis, external oblique, tensor fasciae latae, brachioradialis, iliacus, psoas major, pubococcus, adductor longus, sartorius, gracilis, vastus lateralis, rectus femoris, vastus medialis, quadriceps tendon, patella, gastrocnemius, soleus, tibia, peroneus longus, and tibialis anterior. , patellar ligament, iliotibial band, thenar muscles, 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, anconeus, abductor pollicis longus, plantaris, Achilles tendon, soleus, adductor magnus, gluteus maximus, gluteus medius, latissimus dorsi, infraspinatus, and combinations thereof.

[0311] Administration to smooth muscles Smooth muscles suitable for administration of the compositions of the present invention include any of the walls of blood vessels, stomach walls, ureters, intestinal tract, lining of the aorta (tunica media layer), iris of the eye, prostate, gastrointestinal tract, respiratory tract, small arteries, arterioles, reproductive system (both sexes), veins, glomeruli of the kidney (called mesangial cells), bladder (detrusor muscle), uterus, arrector pili muscles of the skin, ciliary muscles, sphincters (esophagus, anus), trachea, bile duct, etc.

[0312] myocardium The pharmaceutical composition of the present invention is suitable for injection (infusion) into epicardial fat pads (fat pads) such as the sinus node (superior right pulmonary vein (PV), right inferior pulmonary vein) fat pad and the atrioventricular (AV) node (inferior vena cava-left atrium) fat pad.

[0313] secretory gland The pharmaceutical compositions of the present invention can be injected directly into the salivary glands to reduce saliva secretion, into the submandibular gland (under the floor of the mouth) or parotid gland (behind the jaw), or near the eccrine sweat glands in the palms of the hands and feet or in the armpits.

[0314] Repetitive injections Multiple injections and / or injection sites may be necessary to achieve the desired results. The frequency and amount of at least one postsynaptic inhibitor of cholinergic neuronal transmission and botulinum toxin used in the compositions of the present invention can be determined by one of skill in the art based on the nature and location of the particular area to be treated, using, for example, photographs, scans, MRI, electromyograms, etc.

[0315] Injection Procedure The compositions of the present invention can be injected using syringes, catheters, needles, and other injection means. Injections can be made into any area of the body requiring treatment, including, but not limited to, the face, neck, torso, arms, hands, legs, and feet. Injections can be made anywhere in the body, including the epidermis, dermis, subcutaneous layer, fat, or muscle.

[0316] route The route of administration and dosage of the compositions of the present invention can be selected based on criteria such as the solubility characteristics of the postsynaptic inhibitor of cholinergic neuronal transmission and / or the botulinum neurotoxin, and the intensity and extent of the cosmetic or therapeutic condition to be treated.

[0317] Dosage In the injectable composition of the present invention, the useful dosage of at least one postsynaptic inhibitor of cholinergic neuronal transmission used is about 0.01 ng / kg to 500 μg / kg, preferably about 1 to 80 μg / kg, and more preferably about 4 to 50 μg / kg, based on the body weight of the patient to be treated.

[0318] The dosage selection of the at least one postsynaptic inhibitor of cholinergic neuronal transmission may also depend on the LD50 (median lethal dose that kills 50% of a test population) specific to each PoNT. In this case, a useful dosage of the at least one postsynaptic inhibitor of cholinergic neuronal transmission used is about 1% to 50% of the LD50, preferably about 5% to 30% of the LD50, and more preferably about 10% to 20% of the LD50.

[0319] The useful dose range for the botulinum neurotoxin used in the injectable composition of the present invention is about 1 to 1000 units for treatment, preferably about 100 to 500 units, preferably about 50 to 200 units, and more preferably about 20 to 100 units.

[0320] "Unit" refers to the amount of active BoNT normalized to have the same neuromuscular blocking effect as one unit of commercially available botulinum neurotoxin type A. It is well known that units of different BoNT / A products are not equivalently potent and therefore not interchangeable. While all forms of type A toxin have the same mechanism of action, the theoretical number / amount of active 150 kDa molecules in a vial varies depending on the manufactured product, and this variation can have a relative relationship to the LD50 (the median lethal dose that kills 50% of a test population). The LD50, for example, may be expressed in units per mL and is unique to each company's product, defining the potency unit for that product. It may be necessary to calibrate the toxin units against a commercially available reference product.

[0321] In some embodiments, the botulinum neurotoxin can be administered in a total dose of 5 to 1000 U.

[0322] In some embodiments, the botulinum neurotoxin can be administered at a dose of 1 U to 500 U per injection, preferably 10 U to 100 U, and more preferably 20 U to 80 U. Most preferably, the botulinum neurotoxin can be administered at a dose of 20 U to 50 U per injection.

[0323] The dosage of botulinum neurotoxin can also be expressed in terms of protein amount. For example, in certain embodiments, the botulinum neurotoxin can be administered in an amount of about 6 pg to 50 ng, preferably 10 pg to 45 ng, preferably 20 pg to 30 ng, and more preferably 100 pg to 15 ng per dose.

[0324] Dosage form In terms of its form, the composition of the present invention may include a solution, emulsion (including microemulsion or nanoemulsion), suspension, gel, powder, or other typical solid or liquid composition used for administration to muscle and other tissues in which the composition may be used. In a preferred embodiment, the composition of the present invention is present in a low-viscosity sterile formulation suitable for injection with a syringe. As used herein, the terms composition and formulation are essentially interchangeable when referring to the composition and formulation of the present invention. The composition of the present invention may also be in the form of a lyophilized powder that is reconstituted with a pharmaceutically acceptable liquid diluent prior to injection. In addition to at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin, the compositions of the present invention may contain other ingredients commonly used in such products, such as antimicrobial agents, hydrating agents, tissue extenders or fillers, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, antioxidants, fillers, thickeners, powders, viscosity modifiers and water, and optionally anesthetics, antipruritics, plant extracts, conditioning agents, minerals, polyphenols, silicones or derivatives thereof, vitamins, plant-derived medicinal ingredients, etc.

[0325] The injectable compositions of the present invention may be in the form of controlled-release or sustained-release compositions in which at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or postsynaptic small molecule, and a botulinum toxin are encapsulated or otherwise contained within a material for controlled release into tissue over time. The composition containing the botulinum neurotoxin and the postsynaptic inhibitor of cholinergic neuronal transmission may be contained within a matrix, liposome, vesicle, microcapsule, microsphere, or the like, or may be contained within a solid particulate material, all of which are selected and / or constructed to provide time-dependent release of the botulinum toxin. The at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or postsynaptic small molecule, and the botulinum toxin may be encapsulated together (i.e., in the same capsule) or separately (i.e., in separate capsules).

[0326] Topical administration The botulinum toxin formulations of the present invention can be delivered by injection (typically with a syringe) to muscles beneath the skin or to glandular structures within the skin in an amount effective to produce paralysis, produce relaxation, relieve contractions, prevent or relieve spasms, decrease glandular output, relieve pain due to muscle contractions, or other desired effect. Such localized delivery of botulinum toxin results in reduced dosage, reduced toxicity, and allows for more precise optimization of dosage for the desired effect compared to injections or implants.

[0327] Effective dose The compositions of the present invention are administered to deliver an effective amount of botulinum toxin, preferably a therapeutically or cosmetically effective amount. As used herein, the terms "effective amount" or "therapeutically or cosmetically effective amount" refer to an amount of botulinum toxin, as defined above, sufficient to produce the desired muscle relaxation or other biological or cosmetic effect, but implicitly a safe amount, i.e., low enough to avoid serious side effects. Desired effects include, for example, relaxation of specific skeletal muscles to reduce excessive muscle tone (in neuromuscular disorders such as dystonia), reduce the appearance of fine lines and / or wrinkles, especially on the face, or otherwise adjust facial appearance, such as widening the eyes, lifting the corners of the mouth, or smoothing the lines fanning out from the upper lip, or to generally reduce muscle tension. This latter effect of generally reducing muscle tension can be achieved not only on the face but also elsewhere. A further desired effect is related to the relaxation of smooth muscles, the purpose of which is to reduce abnormal contractions. Such abnormal contractions occur, for example, in patients suffering from overactive bladder. Another desirable effect includes reducing nerve cell stimulation of the myocardium. Pathological nerve cell stimulation of myocardial cells is associated with cardiac conditions such as atrial fibrillation, and there is a legitimate need to reduce such stimulation. A secondary desirable effect relates to reducing gland stimulation in pathological conditions such as hyperhidrosis or sialorrhea.

[0328] The compositions of the present invention may contain an effective amount of botulinum toxin suitable for application as a single treatment, or may be more concentrated for dilution at the site of administration or for use in multiple applications. The botulinum toxin can be administered to a subject by injection to treat conditions such as wrinkles, unwanted facial or other muscle spasms, hyperhidrosis, acne, or conditions elsewhere in the body where relief of muscle pain or spasms is desired, through the use of at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or small molecule. The compositions of the present invention are particularly suitable for treating fine lines, such as facial wrinkles, and glabellar lines, also known as "frown lines," on a subject's face. The botulinum toxin is administered by injection into muscles or other skin-related or other effector tissue structures. Administration may be to, for example, the legs, shoulders, back (including the lower back), armpits, palms, feet, neck, face, groin, backs of hands and feet, elbows, upper arms, knees, thighs, buttocks, trunk, pelvis, or any other part of the body where administration of botulinum toxin is desired.

[0329] Indications cosmetic A fourth object of the present invention is a cosmetic composition for use in reducing wrinkles, lines such as glabellar lines or furrows, cosmetic muscle volume (such as masseter or calf muscles), hypertrophic scars, and other dermatological conditions in an individual in need thereof, the cosmetic composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or small molecule, a botulinum toxin, and a cosmetically acceptable diluent for injection.

[0330] therapeutic Administration of the injectable botulinum toxin-containing compositions of the invention may also be carried out to treat other conditions, including any condition in which prevention of synaptic transmission, prevention of acetylcholine release, or prevention of release of other neurotransmitters is believed to provide therapeutic benefit.

[0331] For example, conditions that may be treated with the compositions of the present invention include, but are not limited to, dystonia and spasticity. The compositions of the present invention may also be used to treat other conditions for which administration of botulinum toxin by injection has been proposed or practiced, such as pain associated with muscle contractions, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions (whether subjective or clinical), hyperhidrosis (whether subjective or clinical), and hypersecretion of one or more glands controlled by the cholinergic nervous system.

[0332] A fifth object of the present invention is a pharmaceutical composition for use in the treatment of movement disorders, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the upper limbs, blepharospasm, eyelid disorders, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasm, hemifacial spasm, tremor, tics, bruxism, apraxia and freezing of gait, comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e. a postsynaptic peptide or a postsynaptic small molecule, a botulinum toxin and a pharmaceutically acceptable diluent for injection.

[0333] In the above therapeutic indications, skeletal muscle cells are the postsynaptic cells.

[0334] Another object of the present invention is a pharmaceutical composition for use in the treatment of spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophageal disorders, gastroparesis, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, dysuria, fecal incontinence, constipation, anismus, anal fissures, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus (priapism), and other muscle tone disorders, as well as other disorders characterized by involuntary movements of muscle groups, comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or small molecule, a botulinum toxin, and a pharmaceutically acceptable diluent for injection.

[0335] In the above therapeutic indications, smooth muscle cells are the postsynaptic cells.

[0336] Another object of the present invention is a pharmaceutical composition for use in the treatment of atrial fibrillation, comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, a botulinum toxin, and a pharmaceutically acceptable diluent for injection.

[0337] In the above therapeutic indications, cardiomyocytes are the postsynaptic cells.

[0338] Another object of the present invention is a pharmaceutical composition for use in the treatment of lacrimation, hyperhidrosis (hands, feet and underarms), sialorrhea, excessive salivation, excessive gastrointestinal secretions, excessive sebaceous gland production (and related conditions such as acne) and other secretory disorders, comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or small molecule, a botulinum toxin and a pharmaceutically acceptable diluent for injection.

[0339] In the above therapeutic indications, the secretory cell is the postsynaptic cell.

[0340] In certain embodiments of these intended uses, the at least one postsynaptic inhibitor of cholinergic neuronal transmission is a fast-acting inhibitor.

[0341] In more particular embodiments of these intended uses, the at least one postsynaptic inhibitor is a fast-acting inhibitor and the botulinum toxin is type A, B or E, preferably type A.

[0342] The present invention also relates to a method of treating an individual in need of treatment with an injectable botulinum neurotoxin, comprising administering to a site in the individual in need of treatment a pharmaceutically effective amount of a pharmaceutical composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or a postsynaptic small molecule, and a botulinum neurotoxin to achieve a cosmetic or therapeutic effect. The present invention is preferably suitable for use in treating conditions associated with undesired activity of cholinergic neurons and its potentially associated pain in a subject in need thereof.

[0343] In certain embodiments, the cosmetic effect is the reduction of wrinkles, lines such as glabellar lines, or furrows, reduction of muscle volume (such as the masseter or calf muscles) for cosmetic purposes, reduction of hypertrophic scars, and treatment of other dermatological conditions.

[0344] In another particular embodiment, the therapeutic effect is reduction of movement disorders, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the upper limbs, blepharospasm, eyelid disorders, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasms, hemifacial spasm, tremors, tics, teeth grinding, apraxia, and freezing of gait.

[0345] In another specific embodiment, the therapeutic effect is relief of spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophageal disorders, gastroparesis, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, dysuria, fecal incontinence, constipation, anismus, anal fissures, relief or alleviation of uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus (priapism), other muscle tone disorders, and other disorders characterized by involuntary movement of muscle groups.

[0346] In another particular embodiment, the therapeutic effect is the reduction of atrial fibrillation.

[0347] In another particular embodiment, the therapeutic effect is a reduction in lacrimation, hyperhidrosis (hands, feet, and underarms), drooling, excessive salivation, excessive gastrointestinal secretions, excessive sebaceous gland production (and related conditions such as acne), and other secretory disorders.

[0348] Therapeutic needs include the treatment of conditions in which the activity of specific cell types must be modulated. The compositions of the present invention enable such modulation.

[0349] In another specific embodiment, when the postsynaptic cell is a skeletal muscle cell, the condition to be treated is movement disorder, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the upper limb, blepharospasm, eyelid disorders, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasm, hemifacial spasm, tremor, tics, teeth grinding, apraxia, and freezing of gait.

[0350] In another specific embodiment, when the postsynaptic cell is a smooth muscle cell, the condition to be treated is spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophageal disorders, gastroparesis, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, dysuria, fecal incontinence, constipation, anismus, anal fissures, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus (priapism), other muscle tone disorders and other disorders characterized by involuntary movement of muscle groups.

[0351] In another specific embodiment, when the postsynaptic cell is a cardiomyocyte, the condition to be treated is atrial fibrillation.

[0352] In another specific embodiment, when the postsynaptic cell is a secretory gland cell, the condition to be treated is lacrimation, hyperhidrosis (hands, feet, and underarms), sialorrhea, excessive salivation, excessive gastrointestinal secretions, excessive sebaceous gland production (and related conditions such as acne), and other secretory disorders.

[0353] The present invention relates to a composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin, or a pharmaceutical composition comprising this composition, Movement disorders, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the upper limbs, blepharospasm, eyelid disorders, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasm, hemifacial spasm, tremor, tics, teeth grinding, apraxia and freezing of gait, Spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophageal disorders, gastroparesis, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, dysuria, fecal incontinence, constipation, anismus, anal fissures, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus (priapism), other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, Atrial fibrillation, Lacrimation, hyperhidrosis (hands, feet and underarms), drooling, excessive salivation, excessive gastrointestinal secretions, excessive sebaceous gland production (and related conditions such as acne) and other secretory disorders It also relates to the use of the compound in the manufacture of a medicament for the treatment of

[0354] A final object of the invention relates to a kit for carrying out the method of the invention, comprising a needle and a corresponding syringe, or a smaller needle and a corresponding syringe, preferably a 25-30 gauge needle.

[0355] Those skilled in the art will be able to select the size of the needle depending on the injection site of the composition of the present invention.

[0356] The kit includes receptors for nicotinic acetylcholine receptors (nAChRs, particularly (α1)2β1δε or (α1)2β1δγ), M1, M2, or M3 muscarinic acetylcholine receptors (mAChRs), voltage-gated L-type calcium channels (CaV1.1 and CaV1.2), ryanodine receptors (RyRs), particularly large conductance Ca2+ receptors known as RyR1 present in skeletal muscle and RYR2 present in smooth or cardiac muscle. +Voltage-gated Na release channel + The composition further comprises at least one postsynaptic inhibitor of cholinergic neuronal transmission, i.e., a postsynaptic peptide or small molecule, having binding specificity for voltage-gated sodium (NaV) channels, particularly NaV1.4 and NaV1.5 channels, α1- or β1-adrenergic receptors, and a botulinum neurotoxin, preferably botulinum neurotoxin type A, B, or E. The at least one postsynaptic inhibitor of cholinergic neuronal transmission and the botulinum toxin are mixed together in a vial (lyophilized or liquid), or presented in two chambers for immediate reconstitution before injection, or reconstituted in two separate vials and injected one after the other.

[0357] The following examples are provided to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting its scope. [Example]

[0358] Injectable botulinum neurotoxin composition BoNT / A composition The BoNT / A composition is an injectable formulation containing botulinum neurotoxin type A (BoNT / A). Dried BoNT / A is reconstituted in saline and supplemented with 33% rat serum according to the manufacturer's instructions. Merz Pharmaceuticals' 150 kDa active botulinum toxin A, XEOMIN®, is used for the study. The test dose of BoNT / A is 2.5-5 units / kg, equivalent to approximately 10-20 pg / kg.

[0359] Combination Composition The combination composition is an injectable preparation containing botulinum neurotoxin type A (BoNT / A) and at least one postsynaptic inhibitor of cholinergic neuronal transmission. Depending on the properties of the postsynaptic neuronal transmission inhibitor, different protocols are used to develop the combination composition.

[0360] 1.2.1 Combination Compositions Using Postsynaptic Peptides Dried BoNT / A was reconstituted in saline according to the manufacturer's instructions. Dried postsynaptic peptides were reconstituted in saline supplemented with rat serum and added to the BoNT / A reconstitution solution. Postsynaptic peptides were obtained by solid-phase peptide synthesis or purchased from a supplier (α-bungarotoxin was purchased from Abcam). The doses of postsynaptic peptides used in the experiments ranged from 1 to 350 μg / kg.

[0361] 1.2.2 Combination Compositions Using Postsynaptic Small Molecules Dried BoNT / A was reconstituted with saline according to the manufacturer's instructions. Dried postsynaptic small molecules were reconstituted with saline, water, or water / DMSO, which can be supplemented with rat serum. The postsynaptic small molecules were finally added to the reconstituted BoNT / A solution. Postsynaptic small molecules were purchased from suppliers (pancuronium, dantrolene, amlodipine, nicardipine, verapamil, and diltiazem were purchased from Abcam). The doses of small molecules used in the experiments ranged from 1 to 1200 μg / kg.

[0362] Experimental procedure Digital Abduction Score (DAS) animal Experiments were performed using adult Sprague-Dawley rats (200 g or 400 g) obtained from Charles River, France, Janvier Labs, France, or Envigo, France. Rats were acclimatized in the animal facility with free access to food and water for at least 1 week before use. Five rats were used per condition unless otherwise noted. Other experiments were performed using CD-1 mice (20 g) obtained and treated as described above.

[0363] Animal weights are recorded on days 0, 2, 4, 7 and weekly throughout the study until the end of the experiment.

[0364] treatment Animals were injected (10 μL or 20 μL per rat) using a 30- or 33-gauge needle attached to a 100 μL Hamilton syringe. On the first day of the experiment, rodents were pre-screened for a "zero" DAS response before injection. Rats then received an intramuscular (IM) injection into the right tibialis anterior (TA) muscle. As a control, the left TA muscle was injected with saline (10 μL).

[0365] Digital Abduction Score Assay (DAS) Muscle paralysis was measured using the digital abduction score (DAS) assay, as reported by Broide et al. (TOXICON, 2013, 71, pp. 18-24). Animals were induced to abduct their digits and scored for DAS responses at different time points after compound injection: 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, 12 h, 24 h, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, and once daily until two consecutive measurements on two consecutive days reached 0 (DAS = 0). Two independent observers blinded to the treatment scored the degree of digital abduction on a 5-point scale (0 = normal to 4 = maximal reduction in digital abduction, i.e., no toes abducted).

[0366] As is common in rat DAS testing, the DAS response was elicited by grasping the rat around the torso, lifting it into the air, and simulating a drop onto a flat surface. The rat typically reflexively spreads its hind limbs in preparation for the impact, and the DAS response was immediately scored with the animal reclined and facing up.

[0367] Data analysis As mentioned above, if there was loss of abduction of the first toe, the DAS score was given a "1." If three toes were united, the DAS score was given a "2." If four toes were united, the score was given a "3." If all five toes on the right paw were united during abduction, the rat was given a score of "4."

[0368] DAS responses at each time point are measured and kinetics (onset of muscle weakness and duration of effect) are assessed and compared in different groups of rats.

[0369] For each rat, the area under the DAS curve (units of DAS × time) was calculated using a series of Riemann sums between T = 0 to T = 24 h (early AUC), T = 24 to T = 120 h (mid-term AUC), and T = 120 h to the end of the experiment (late AUC).

[0370] For each group, the onset of effect was defined as the first time point at which the mean DAS value exceeded 1, the duration of effect was defined as the last time point at which the mean DAS value exceeded 1, and the peak of effect was defined as the maximum mean DAS value.

[0371] Combination effects of BoNT / A and postsynaptic peptides that bind to the voltage-gated sodium channel NaV1.4 To determine the pharmacodynamic profile of the combination of BoNT / A and μ-conotoxin CnIIIc postsynaptic peptide compared to BoNT / A alone, rats were injected into the right tibialis anterior (TA) muscle with 5 U / kg of BoNT / A combined with doses ranging from 0.4 to 40 μg / kg of μ-conotoxin CnIIIc postsynaptic peptide, or with 5 U / kg of BoNT / A alone. The sequence of the μ-conotoxin CnIIIc postsynaptic peptide used in this experiment is SEQ ID NO: 54.

[0372] The results in Figure 1 show that the onset of action of BoNT / A is 12 hours after injection, with a peak effect at 72 hours. The duration of action is 5 days.

[0373] Faster onset of action Injection of the combination of BoNT / A and the mu-conotoxin CnIIIc postsynaptic peptide resulted in a significantly faster onset of action than BoNT / A alone, with the onset of action depending on the dose of mu-conotoxin CnIIIc: slightly after 1 hour at 40 μg / kg, slightly before 2 hours at 20 μg / kg, and around 4 hours at 8 μg / kg.

[0374] Peak Rise The peak effect increases from 3.4 with BoNT / A alone to 4.0 in the presence of the highest dose of mu-conotoxin CnIIIc.

[0375] Increased duration The duration of action of these combinations is also longer than that of BoNT / A alone, depending on the dose of μ-conotoxin CnIIIc used: 168-192 hours at 8 μg / kg, 192-216 hours at 20 μg / kg, and 216-240 hours at 40 μg / kg (compared to just over 120 hours with BoNT / A alone).

[0376] Taken together, these data indicate that the combination of BoNT / A and μ-conotoxin CnIIIc results in a more rapid onset, more potent, and longer-lasting local muscle relaxant effect compared with BoNT / A alone.

[0377] Synergy The enhanced efficacy of BoNT / A observed when combining BoNT / A with μ-conotoxin CnIIIc cannot be explained by a simple additive effect of both components of the composition. These results are shown in Figure 2.

[0378] To analyze the synergistic effect of the combination of BoNT / A and μ-conotoxin CnIIIc postsynaptic peptide, this combination was compared with BoNT / A alone and μ-conotoxin CnIIIc peptide alone. Groups of five rats were injected into the right tibialis anterior (TA) muscle with 5 U / kg of BoNT / A combined with 80 μg / kg of μ-conotoxin CnIIIc postsynaptic peptide, 5 U / kg of BoNT / A alone, or 80 μg / kg of μ-conotoxin CnIIIc postsynaptic peptide. The sequence of the μ-conotoxin CnIIIc postsynaptic peptide used in this experiment is SEQ ID NO: 54.

[0379] The results shown in Figure 2 indicate that the DAS peak for BoNT / A appeared 12 to 24 hours after injection, with a maximum mean score of 2.8 achieved 72 hours after injection. The DAS for BoNT / A began to decline on day 4. The sustained effect was observed up to day 6 after BoNT / A injection. Expression of μ-conotoxin CnIIIc postsynaptic peptide was observed 0.5 to 1 hour after injection, with a maximum intensity of 3.6 achieved 2 hours after injection. The DAS for μ-conotoxin CnIIIc postsynaptic peptide alone began to decline as early as 6 hours after injection. Logically, the calculated sum of both treatments showed two peaks, the first at 2 hours after injection and the second at 72 hours after injection. Noteworthy is that the score between the two peaks returns to a low level (DAS < 2). Surprisingly, the combination of BoNT / A and μ-conotoxin CnIIIc did not show two peaks, but rather produced sustained muscle relaxation from 2-6 hours (onset) to 144-168 hours (duration) after injection. The combination of BoNT / A and postsynaptic μ-conotoxin CnIIIc exhibited a synergistic effect that was greater than the sum of the effects of both components.

[0380] The area under the curve (AUC, units: DAS x time) can be calculated to better understand the synergistic effect.

[0381] [Table 11]

[0382] This table reveals that the effect of the combination of BoNT / A and μ-conotoxin CnIIIc is greater than the sum of the effects of both components with respect to onset (early: 123.4 > 100.8), intensity (mid-phase: 240 > 180.3), and duration (late: 103.2 > 3) of action.

[0383] Combination effects of BoNT / A and postsynaptic inhibitors of cholinergic neuronal transmission that bind to nicotinic acetylcholine receptors (nAChRs) This example compares the onset, intensity, and duration of local muscle relaxation in rats injected with BoNT / A or a combination of BoNT / A and three postsynaptic inhibitors of neuronal transmission that all bind to nAChRs. Only one rat per group was treated.

[0384] Pancuronium To determine the onset and duration of action of BoNT / A compared with the combination of BoNT / A and pancuronium, rats were injected with 5 U / kg BoNT / A or a combination of 5 U / kg BoNT / A and 57 μg / kg pancuronium.

[0385] The results shown in Figure 3 show that the peak DAS score for BoNT / A appeared 12 to 24 hours after injection, with a maximum score of 3.2 achieved after 72 hours. The DAS score for BoNT / A began to decline on day 4. By day 7 after BoNT / A injection, finger abduction was fully restored. Injection of the combination of BoNT / A and pancuronium resulted in a faster onset of the DAS than injection with BoNT / A alone, occurring 5 to 15 minutes after injection. In the presence of pancuronium, the maximum DAS score also increased to 4.0. Finally, the duration of muscle relaxation was slightly increased by adding pancuronium to BoNT / A. Six days after injection, finger abduction was still impaired (DAS = 2), whereas in rats treated with BoNT / A, it had begun to recover (DAS < 1).

[0386] These data indicate that the combination of BoNT / A and pancuronium provides a more potent, more rapid onset of local muscle relaxant and a longer duration of action than BoNT / A alone.

[0387] α-conotoxin postsynaptic peptides To determine the onset, intensity, and duration of action of BoNT / A compared to a combination of BoNT / A and sweet potato (Conus magus) postsynaptic peptide, rats were injected with 5 U / kg BoNT / A or a combination of 5 U / kg BoNT / A and 8 μg / kg α-conotoxin MI peptide of SEQ ID NO: 8.

[0388] The results shown in Figure 3 indicate that the peak DAS score for BoNT / A appeared between 12 and 24 hours after injection, with a maximum score of 3.2 achieved 72 hours after injection. The DAS score for BoNT / A began to decline on day 4. Finger abduction fully recovered by day 7 after BoNT / A injection. Injection of the combination of BoNT / A and α-conotoxin MI peptide resulted in a faster onset of the DAS than injection with BoNT / A alone, occurring between 2 and 4 hours after injection. In the presence of α-conotoxin MI peptide, the maximum DAS score also increased to 4.0. Finally, the duration of muscle relaxation was significantly prolonged by adding α-conotoxin MI peptide to BoNT / A, with finger abduction still not recovered 12 days after injection.

[0389] These data indicate that the combination of BoNT / A and α-conotoxin MI peptide results in a more potent, more localized muscle relaxant with a faster onset and a longer duration of action than BoNT / A alone.

[0390] α-bungarotoxin To determine the onset and duration of action of BoNT / A compared to the combination of BoNT / A and Bungarus toxin peptide, rats were injected with 5 U / kg BoNT / A or a combination of 5 U / kg BoNT / A and 17 μg / kg α-bungarotoxin peptide of SEQ ID NO: 4.

[0391] For each rat, DAS was recorded at several time points, from the first 10 minutes after injection to 12 days (288 hours). The results, shown in Figure 3, show that the peak DAS for BoNT / A occurred between 12 and 24 hours after injection, with a maximum score of 3.2 achieved 72 hours after injection. The DAS for BoNT / A began to decline on day 4. By day 7 after BoNT / A injection, finger abduction was fully restored. Injection of the combination of BoNT / A and α-bungarotoxin resulted in a faster onset of DAS than injection with BoNT / A alone, occurring between 15 and 30 minutes after injection. In the presence of α-bungarotoxin, the maximum DAS also increased to 4.0. Finally, the duration of muscle relaxation was extended by 4 days by adding α-bungarotoxin to BoNT / A.

[0392] These data indicate that the combination of BoNT / A and α-bungarotoxin results in a more rapid onset of local muscle relaxation, a more potent muscle relaxant effect, and a longer duration of action than BoNT / A alone.

[0393] The overall data indicate that the use of postsynaptic inhibitors of neuronal transmission that target nAChRs may result in a faster onset and / or a longer duration of effect and / or increased efficacy of botulinum toxin.

[0394] Combination effects of BoNT / A and postsynaptic inhibitors of cholinergic neuronal transmission that bind to ryanodine receptor (RYR1) Dantrolene To determine the onset and duration of action of BoNT / A compared to the combination of BoNT / A with dantrolene, a known inhibitor of RYR1, rats were injected with 5 U / kg BoNT / A or 5 U / kg BoNT / A combined with 1.6 μg / kg dantrolene.

[0395] For each rat, DAS was recorded at several time points, from the first 10 minutes after injection to 12 days (288 hours). The results, shown in Figure 4, show that the peak DAS for BoNT / A occurred between 12 and 24 hours after injection, with a maximum score of 3.2 achieved 72 hours after injection. The DAS for BoNT / A began to decline on day 4. By day 7 after BoNT / A injection, finger abduction was fully restored. Injection of the combination of BoNT / A and dantrolene resulted in a more rapid onset of DAS than injection with BoNT / A alone, occurring 2 to 4 hours after injection. In the presence of dantrolene, the maximum DAS also increased to 4.0. Finally, the duration of muscle relaxation was significantly delayed by the addition of dantrolene to BoNT / A, with finger abduction still not restored 12 days after injection.

[0396] These data indicate that the combination of BoNT / A and dantrolene provides a more rapid onset of local muscle relaxation, a more potent muscle relaxant effect, and a longer duration of action than BoNT / A alone.

[0397] Insecticidal toxin LaIT1 To determine the onset and duration of action of BoNT / A compared to the combination of BoNT / A and the insecticidal toxin LaIT1, rats were injected with 5 U / kg of BoNT / A or 5 U / kg of BoNT / A and 36 μg / kg of the LaIT1 peptide of sequence number 23.

[0398] For each rat, DAS was recorded at several time points, from the first 10 minutes to 12 days (288 hours) after injection. The results, shown in Figure 4, indicate that the peak DAS score for BoNT / A occurred between 12 and 24 hours after injection, with a maximum score of 3.2 achieved 72 hours after injection. The DAS for BoNT / A began to decline on day 4. By day 7 after BoNT / A injection, finger abduction was fully restored. Injection of the combination of BoNT / A and the insecticidal toxin LaIT1 resulted in a faster onset of DAS than injection with BoNT / A alone, with onset occurring between 2 and 6 hours after injection. In the presence of the insecticidal toxin LaIT1, the maximum DAS also increased to 4.0. Finally, the duration of muscle relaxation was delayed by 5 days by adding the insecticidal toxin LaIT1 to BoNT / A. Thus, a longer duration of action of BoNT / A was achieved.

[0399] These data indicate that the combination of BoNT / A and the insecticidal toxin LaIT1 results in a more potent, localized muscle relaxant with a faster onset and a longer duration of action than BoNT / A.

[0400] The overall data indicate that the use of postsynaptic inhibitors of neuronal transmission that target RYR1 may result in a faster onset and / or a longer duration of effect and / or increased efficacy of botulinum toxin.

[0401] Combination effects of BoNT / A and two closely related postsynaptic inhibitors of cholinergic neuronal transmission that bind to the voltage-gated sodium channel NaV1.4 The pharmacodynamic profile of the combination of BoNT / A and the μ-conotoxin CnIIIc postsynaptic peptide was compared with that of the combination of BoNT / A and a mutant μ-conotoxin CnIIIc peptide. The mutant peptide used in this experiment had a sequence (SEQ ID NO: 95) consistent with the consensus sequence defined in SEQ ID NO: 53, and the sequence of the wild-type μ-conotoxin CnIIIc peptide used in this experiment was SEQ ID NO: 54. For this comparison, rats were injected into the right tibialis anterior (TA) muscle with a combination of 5 U / kg of BoNT / A and 25 μg / kg of each μ-conotoxin CnIIIc postsynaptic peptide. Control rats were injected with 5 U / kg of BoNT / A alone.

[0402] The results were analyzed by calculating the area under the curve (AUC) for the early, middle, and late phases of the effect. For each phase, the percentage increase in AUC relative to the control (BoNT / A alone) is presented for comparison. The final calculation results, shown in Figure 5, firstly show that both combinations can increase the onset of action (early AUC), the intensity of action (middle AUC), and the duration of action (late AUC), all of which are greater than 0%. Secondly, the above results show that the combination with the mutant exhibits a slightly different pharmacological profile compared to the combination with wild-type μ-conotoxin CnIIIc. The former's effect is more pronounced in the early and late phases, with a slightly smaller impact on peak intensity.

[0403] Overall, the data demonstrate the advantage of classifying postsynaptic peptide inhibitors into families that share a consensus sequence: on the one hand, peptides within a family share similar effects, but on the other hand, their slightly different pharmacological profiles can be advantageously exploited in drug design.

[0404] Combination effects of BoNT / A and postsynaptic inhibitors of cholinergic neuronal transmission that bind to voltage-gated L-type calcium channels (Cav1.1) Amlodipine To determine the onset and duration of action of BoNT / A compared to the combination of BoNT / A and amlodipine, a known inhibitor of Cav1.1, rats were injected with 5 U / kg BoNT / A or 5 U / kg BoNT / A in combination with 83 μg / kg amlodipine.

[0405] For each rat, DAS was recorded at several time points, from the first 10 minutes after injection until 14 days (336 hours). The results, shown in Figure 6, indicate that the peak DAS for BoNT / A occurred between 24 and 48 hours after injection, with a maximum score of 3.8 achieved at 72 hours after injection. The DAS for BoNT / A began to decline on day 4, but remained above 1 until day 11. Injection of the combination of BoNT / A and amlodipine resulted in a more rapid onset of DAS than injection with BoNT / A alone, occurring between 2 and 4 hours after injection. In the presence of amlodipine, the maximum DAS also increased to 4.0. Finally, the duration of muscle relaxation was delayed by adding amlodipine to BoNT / A, with finger abduction scores remaining above 1 until day 13.

[0406] These data indicate that the combination of BoNT / A and amlodipine provides a more potent, more rapid onset of local muscle relaxant and a longer duration of action than BoNT / A alone.

[0407] Diltiazem To determine the onset and duration of action of BoNT / A compared with the combination of BoNT / A and diltiazem, a known inhibitor of Cav1.1, rats were injected with 5 U / kg BoNT / A or 5 U / kg BoNT / A in combination with 83 μg / kg diltiazem.

[0408] For each rat, DAS was recorded at several time points, from the first 10 minutes after injection until 14 days (336 hours). The results, shown in Figure 6, show that the peak DAS for BoNT / A occurred between 24 and 48 hours after injection, with a maximum score of 3.8 achieved 72 hours after injection. The DAS for BoNT / A began to decline on day 4, but remained above 1 until day 11. Injection of the combination of BoNT / A and diltiazem resulted in a more rapid onset of DAS compared with injection with BoNT / A alone, occurring between 0.5 and 1 hour after injection. In the presence of diltiazem, the maximum DAS also increased to 4.0. Finally, the duration of muscle relaxation was delayed by adding diltiazem to BoNT / A, with finger abduction scores remaining above 1 until day 13.

[0409] These data indicate that the combination of BoNT / A and diltiazem provides a more potent, more rapid onset of local muscle relaxant and a longer duration of action than BoNT / A alone.

[0410] Verapamil To determine the onset and duration of action of BoNT / A compared to the combination of BoNT / A with verapamil, a known inhibitor of Cav1.1, rats were injected with 5 U / kg BoNT / A or 5 U / kg BoNT / A combined with 83 μg / kg verapamil.

[0411] For each rat, DAS was recorded at several time points, from the first 10 minutes after injection to 14 days (336 hours). The results, shown in Figure 6, indicate that the peak DAS for BoNT / A occurred 24–48 hours after injection, with a maximum score of 3.8 achieved 72 hours after injection. The DAS for BoNT / A began to decline on day 4, but remained above 1 until day 11. Injection of the combination of BoNT / A and verapamil resulted in a more rapid onset of DAS than injection with BoNT / A alone, occurring 4–6 hours after injection. In the presence of verapamil, the maximum DAS also increased to 4.0. Finally, the duration of muscle relaxation was comparable to that of BoNT-induced muscle relaxation.

[0412] These data indicate that the combination of BoNT / A and verapamil provides a more potent and more rapid onset of local muscle relaxation than BoNT / A alone.

[0413] Combination effects of BoNT / A and small molecules that are postsynaptic inhibitors of cholinergic neuronal transmission: A comparison To determine the pharmacological profile of BoNT / A compared with the combination of BoNT / A and small molecules that act as postsynaptic inhibitors, rats were injected with 5 U / kg BoNT / A or 5 U / kg BoNT / A combined with various doses of several small molecules. These molecules were known inhibitors of nAChRs (pancuronium, suxamethonium), RYR1 (dantrolene), or Cav1.1 (amlodipine, diltiazem, verapamil). Results were analyzed by calculating the area under the curve (AUC) over the course of the experiment. For comparison, the percent increase in AUC relative to the control (BoNT / A alone) is presented.

[0414] The results shown in Figure 7 indicate that all small molecules tested can enhance the muscle relaxant effect of BoNT, with the combined effect of dantrolene and BoNT being particularly pronounced.

[0415] Effects of the combination of BoNT / A and peptides, postsynaptic inhibitors of cholinergic neuronal transmission: A comparison To determine the pharmacological profile of BoNT / A compared with combinations of BoNT / A with postsynaptic inhibitor peptides, rats were injected with 5 U / kg BoNT / A or 5 U / kg BoNT / A combined with various doses of several peptides. These molecules were known inhibitors of nAChRs (α-bungarotoxin, α-conotoxin MI, waglerin-1, αC-conotoxin PrXA), RYR1 (LaIT1 toxin, impellacarcin), Cav1.1 (U7-ctenitoxin Pn1b, ω-conotoxin TxVII, U6-ctenitoxin Pn1a, U9-ctenitoxin Pn1a, κ-ctenitoxin Pn1a, and glaucontryphan), or Nav1.4 (μ-conotoxin CnIIIc, μ-conotoxin GIIIb, μ-tomitoxin Hme1a, μ-conotoxin GvIIJ, μO-conotoxin MfVIA, and μ-tomitoxin Hme1b). Results were analyzed by calculating the area under the curve (AUC) over the course of the experiment. For comparison, the % increase in AUC relative to the control (BoNT / A alone) is presented.

[0416] The results shown in Figure 8 indicate that all tested small molecules can enhance the muscle relaxant effect of BoNT, with the combination of α-conotoxin MI and BoNT being particularly effective.

[0417] ·Compound muscle action potential (CMAP) animal Experiments were performed using adult Sprague-Dawley rats (200 g) obtained from Charles River, France, Janvier Labs, France, or Envigo, France. Rats were acclimated to the animal facility with free access to food and water for at least one week before use.

[0418] Animal weights were recorded daily throughout the study until the end of the experiment.

[0419] Data Acquisition and Processing ·treatment Animals were injected (20 μL per rat) using a 33-gauge needle attached to a 100 μL Hamilton syringe, followed by an intramuscular (IM) injection into the right tibialis anterior (TA) muscle.

[0420] ·anesthesia For each CMAP measurement, animals are anesthetized with isoflurane. Prior to anesthesia, animals are kept on a heating plate at 37°C to control body temperature and hydrated with saline (subcutaneous injection).

[0421] ·measurement For the measurement, place the animal in a prone position on a heating pad. Place the electrode needle for CMAP measurement as follows: · Place sham electrodes subcutaneously on either side of the sciatic notch. Place the recording electrode subcutaneously along the tibialis anterior muscle. Place a reference electrode subcutaneously next to the Achilles tendon. A ground electrode is placed subcutaneously opposite the sham electrode.

[0422] CMAP measurements are performed and recorded by stimulation using a Natus® UltraPro S100 EMG device.

[0423] Typically, this measurement is performed 6 days before injection for a baseline measurement and at different time points after compound injection (2 hours, 1 day, 2 days, 4 days, 7 days, 9 days, 11 days, and 14 days).

[0424] Data analysis During the measurement, two parameters are analyzed: latency and peak-to-peak amplitude. Latency is determined by the delay between stimulation and the onset of the CMAP response. Peak-to-peak amplitude is measured from the maximum negative peak to the maximum positive peak of the biphasic wave. The CMAP amplitude can then be plotted as a function of measurement time. In this curve, the onset of action is defined as the time point at which inhibition is greater than 20% of baseline, the peak of activity is defined as the maximum inhibition (percentage of baseline), and the duration of action is defined as the time required to recover to 20% of baseline.

[0425] All data are compared by group using Student's t-test and compared for homogeneity using Fisher's test.

[0426] In vivo model of neurogenic detrusor overactivity animal Experiments were performed using adult Sprague-Dawley rats (200 g) obtained from Charles River, France, Janvier Labs, France, or Envigo, France. Rats were acclimated to the animal facility with free access to food and water for at least one week prior to use. After this period, animals underwent a T8-T9 spinal cord resection. Animal weights were recorded daily throughout the study until the end of the experiment.

[0427] treatment On day 19 after spinal cord transection, when neurogenic detrusor overactivity was established, the animals were anesthetized with isoflurane, and the bladder was exposed and emptied. Using a microscope, the detrusor was injected using a 30G needle attached to a Hamilton syringe containing the solution. The injected volume was distributed over four or eight injection sites, avoiding the trigone of the bladder. A catheter was then inserted into the dome of the bladder, tunneled subcutaneously, externalized behind the neck, and sutured between the scapulae. Postoperatively, the rats were treated with gentamicin.

[0428] measurement Cystometry of rats was performed at 24 hours, 48 hours, 7 days, and 14 days after intradetrusor injection.

[0429] First, micturition and filling parameters were analyzed: maximum pressure of micturition contractions (millimeters of mercury mmHg), volume infused (bladder capacity: μL), and micturition efficiency (ratio of micturition volume to infused volume %). Second, non-micturition contractions (contractions with amplitudes greater than 3 mmHg during the filling phase) were analyzed: amplitude (mmHg), frequency (number of times per minute), and volume threshold (% of total filling volume) to induce the non-micturition phase.

[0430] Data analysis All data were expressed as mean ± standard error of the mean for each treatment group. Grubb's test was used to exclude outliers. Analysis of variance was performed to compare groups for each cystometry parameter, allowing for aggregation.

[0431] Ex vivo bladder model animal Experiments were performed using adult Sprague-Dawley rats (200 g) obtained from Charles River, France, Janvier Labs, France, or Envigo, France. Rats were acclimated to the animal facility with free access to food and water for at least 1 week prior to use. On the day of the experiment, rats were anesthetized with isoflurane and exsanguinated prior to tissue collection. The bladders were harvested and washed. Two approximately 6 x 2 mm strips were excised from each bladder. These were then fixed in a custom electrode tissue holder and allowed to contract to 0.5 g at 37°C, pH 7.4, in an organ bath typically filled with KHB and bubbled with carbogen. Bladder contraction force was measured using an isometric transducer. After an equilibration period of approximately 45 min, the buffer was refreshed every 15 min, and KCl (typically 70 mmol / L) was added to maintain bladder integrity. A solution of carbachol (which activates acetylcholine receptors) is applied to the postsynaptic receptor under test (usually 10 μmol / L), and then contractions of the detrusor muscle are induced by electrical field stimulation using a train of pulses. This stimulation is generated using two platinum electrodes placed a few millimeters on either side of the strip.

[0432] treatment After a period of extensive washing and stable contraction, treatment solutions were added to the bath. Treatment solutions contained various concentrations of BoNT / A, either alone or mixed with postsynaptic inhibitors. Each strip was exposed to only one treatment solution, and the signal was recorded for 6 hours until 90% of the signal had disappeared, or if this value was not reached, for 6 hours. At the end of the experiment, a final addition of carbachol was made to assess tissue viability. If the post-stimulation carbachol response was less than 80% of the pre-stimulation carbachol response, the experiment was rejected.

[0433] Data analysis Results are expressed as a percentage of the initial control contraction immediately before treatment. Data are presented as individual data or as mean ± standard error of the mean. Concentration curves were plotted for each group of conditions. Statistical analysis was performed using an unpaired Student's t-test.

Claims

1. A method for enhancing the effect of a botulinum neurotoxin composition, comprising the step of adding at least one postsynaptic inhibitor of cholinergic neuronal transmission to said botulinum neurotoxin composition.

2. The method of claim 1, wherein enhancing the effect of the botulinum neurotoxin composition comprises accelerating the onset of action of the botulinum neurotoxin composition, and / or extending the duration of action, and / or increasing the intensity of action.

3. 3. The method of claim 1, wherein the onset of action of the botulinum neurotoxin composition comprising the at least one postsynaptic inhibitor of cholinergic neuronal transmission occurs 50%, preferably 75%, more preferably 90% faster than the onset of action of a botulinum neurotoxin composition not comprising a postsynaptic inhibitor of cholinergic neuronal transmission.

4. 4. The method of claim 1, wherein the duration of action of the botulinum neurotoxin composition comprising the at least one postsynaptic inhibitor of cholinergic neuronal transmission is extended by 10%, 25%, 50%, 100% or more compared to the duration of action of a botulinum neurotoxin composition not comprising a postsynaptic inhibitor of cholinergic neuronal transmission.

5. 5. The method according to claim 1, wherein the potency of the action of the botulinum neurotoxin composition comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission is increased by at least 2%, preferably at least 5%, more preferably at least 10% compared to the maximum potency of the action of a botulinum neurotoxin composition not comprising a postsynaptic inhibitor of cholinergic neuronal transmission.

6. 6. A composition suitable for carrying out the method of any one of claims 1 to 5, comprising at least one postsynaptic inhibitor of cholinergic neuronal transmission and a botulinum neurotoxin.

7. The at least one postsynaptic inhibitor of cholinergic neuronal transmission specifically binds to at least one receptor expressed by skeletal muscle cells, the receptor being selected from the group consisting of (α1) ２ β1δε nAChr, (α1) ２ 7. The composition of claim 6, comprising or selected from the group consisting of β1δγ nAChr, RyR1, CaV1.1 and NaV1.

4.

8. 7. The composition of claim 6, wherein the at least one postsynaptic inhibitor of cholinergic neuronal transmission specifically binds to at least one receptor expressed by smooth muscle cells selected from the group including or consisting of M3 mAchr, RyR2, CaV1.2, and NaV1.

5.

9. 7. The composition of claim 6, wherein the at least one postsynaptic inhibitor of cholinergic neuronal transmission specifically binds to at least one receptor expressed by cardiac myocytes, the receptor comprising or selected from the group consisting of M2 mAchr, RyR2, CaV1.1, CaV1.2, and NaV1.

5.

10. The at least one postsynaptic inhibitor of cholinergic neuronal transmission specifically binds to at least one receptor expressed by secretory gland cells, the receptor being selected from the group consisting of M1 mAChR, M3 mAChR, α １ 7. The composition of claim 6, comprising or selected from the group consisting of adrenoceptors and β1 adrenoceptors.

11. 11. The composition of any one of claims 6 to 10, wherein the at least one postsynaptic inhibitor of cholinergic neuronal transmission is a postsynaptic peptide and / or a postsynaptic small molecule.

12. 12. The composition of any one of claims 6 to 11, wherein the at least one postsynaptic inhibitor of cholinergic neuronal transmission is α-conotoxin MI, μ-conotoxin CnIIIc, or a derivative thereof, pancuronium, dantrolene, or a combination thereof.

13. 13. The composition of any one of claims 6 to 12, wherein the at least one postsynaptic inhibitor of cholinergic neuronal transmission is a fast-acting postsynaptic peptide and / or a fast-acting postsynaptic small molecule.

14. 13. The composition of any one of claims 6 to 12, wherein the at least one postsynaptic inhibitor of cholinergic neuronal transmission is compatible with the delayed action of a botulinum neurotoxin.

15. 15. The composition of any one of claims 6 to 14, wherein the botulinum neurotoxin is a type A, a type B, a type E, or a combination of the heavy and light chains of a type A, a type B, or a type E botulinum neurotoxin.

16. 16. A composition according to any one of claims 6 to 15 for use in cosmetic treatment such as reducing wrinkles, lines such as glabellar lines or grooves, muscle volume (such as masseter or calf muscles) for cosmetic purposes, hypertrophic scars and other dermatological conditions.

17. 16. A composition according to any one of claims 6 to 15 for use in the treatment of (i) musculoskeletal disorders including movement disorders, dystonia, cervical dystonia, spasmodic torticollis, focal dystonia, focal dystonia of the upper limb, blepharospasm, eyelid disorders, strabismus, spasticity, cerebral palsy, focal spasticity, limb spasticity, spasms, hemifacial spasm, tremor, tics, teeth grinding, apraxia and freezing of gait, and (ii) pain associated with these disorders.

18. 16. A composition according to any one of claims 6 to 15 for use in the treatment of (i) smooth muscle disorders including spasmodic dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual dystonia and other voice disorders, achalasia, dysphagia, esophageal disorders, gastroparesis, spastic colitis, neurogenic bladder, overactive bladder, interstitial cystitis, benign prostatic hyperplasia, dysuria, fecal incontinence, constipation, anismus, anal fissures, uterine pain (dysmenorrhea, dyspareunia), vaginal pain (vaginismus, vulvodynia), pelvic pain, ischiocavernosus (priapism), other muscle tone disorders and other disorders characterized by involuntary movement of muscle groups, and (ii) pain associated with these disorders.

19. 16. A composition according to any one of claims 6 to 15 for use in the treatment of cardiomyocyte damage, including atrial fibrillation.

20. 16. A composition according to any one of claims 6 to 15 for use in the treatment of glandular disorders including lacrimation, hyperhidrosis of the hands, feet and underarms, sialorrhea, excessive salivation, excessive gastrointestinal secretions, excessive sebaceous gland production, acne and secretory disorders.

21. 21. A composition according to any one of claims 6 to 15 for use as defined in any one of claims 16 to 20, which has an accelerated onset of action and / or a prolonged duration of action and / or an enhanced intensity of action compared to a botulinum neurotoxin composition that does not contain a postsynaptic inhibitor of cholinergic neuronal transmission.

22. 7. The composition of claim 6, wherein the at least one postsynaptic inhibitor of cholinergic neuronal transmission (PoNT) and the botulinum neurotoxin are administered in combination, provided that the botulinum neurotoxin is administered before attenuation of PoNT activity occurs.

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