Topical pain-relief pharmaceutical composition comprising an omega-conotoxin

Abstract

The invention relates to a topcially-acting pain-relief pharmaceutical composition for topical use, comprising, as an active ingredient, one or more natural or synthetic peptides from the ω-conotoxin family, or one or more functionally active natural or synthetic variants thereof, the composition being intended for use in a method for treating acute or chronic nociceptive, neuropathic or nociplastic pain, such as intense chronic inflammatory and neuropathic pain, and being characterised in that it is administered locally, via the skin, close to and at the site of the free endings in the skin of the nociceptors that innervate the skin.

Description

Description TOPICAL PAIN RELIEVER PHARMACEUTICAL COMPOSITION COMPRISING AN OMEGA-CONOTOXIN

[0001] The present invention falls within the field of pharmaceutical compositions, comprising as active ingredient one or more peptide(s) from the co-conotoxin family, for use in the treatment of acute or chronic pain, of a nociceptive, neuropathic or nociplastic nature, such as for example intense chronic inflammatory and neuropathic pain.

[0002] The invention particularly relates to a pharmaceutical composition comprising a co-conotoxin, for example of the MVIIA type, for use in a method of treating severe chronic inflammatory and neuropathic pain, which is administered locally, via the skin, near the sensory fiber endings of cutaneous nociceptors innervating the skin.

[0003] Co-conotoxins are toxins produced either naturally by marine molluscs of the genus Conus, or synthetically by any peptide synthesis methods known to those skilled in the art.

[0004] Naturally occurring co-conotoxins identified in marine cones such as Conus cactus, Conus magus, Conus geographus, Conus slrialus, Conus tulipa, Conus consors, Conus textile, Conus radiatus, and Conus fulmen are typically peptides with 10 to 30 amino acid residues, which may have post-translational modifications such as an amidated C-terminus and up to six cysteine ​​residues arranged to form a three-dimensional four-loop structure.

[0005] Co-conotoxins act on so-called "voltage-gated calcium channels" (Cav), particularly including N-type voltage-gated calcium channels abundant in the dorsal horn region of the spinal cord.

[0006] Cavs help transmit the nociceptive message from the peripheral nervous system to the central nervous system. Cavs are one of the main pathways for calcium entry into the nerve cell in response to an action potential, which involves the release of neurotransmitters into the synapse, and consequently, the transmission of the pain signal. Cavs are therefore key players in the transmission of the nociceptive signal (Hoppanova, L et al, (2022) Pflugers Arch - Eur J Physiol 474, 421-434, DOI 10.1007 / s00424-022-02666-y) and a prime pharmacological target for analgesics.

[0007] Consequently, co-conotoxins, targeting N-type Cavs, involved in the synaptic transmission of the nociceptive signal, are of great therapeutic interest, particularly as an alternative to opiates in the treatment of pain.

[0008] However, peptides of the co-conotoxin family, which impact Cavs, are peptides potentially sensitive to proteases and peptidases. This sensitivity limits their bioavailability in the body and complicates their administration in order to act on a defined therapeutic target.

[0009] In order to increase their stability in vivo, in particular to avoid alteration by proteases, or to facilitate their distribution, it has been considered to synthesize modified co-conotoxins, such as for example cyclic co-conotoxins, comprising modified amino acids or coupled with protective groups. However, this synthesis of modified peptides is complex because a modification of the three-dimensional structure of the peptide or its physicochemical characteristics (charge, polarity, molecular weights) does not guarantee the conservation of its biological activity. In particular, modifying the biochemical structure of a peptide of the co-conotoxin family can complicate its administration, impact its selectivity and specificity of action on Cavs or even increase its toxicity towards the organism.

[0010] Still with the aim of using co-conotoxins in therapy, as an alternative to opiates which can cause addiction, it has been considered to administer co-conotoxins directly and as close as possible to the therapeutic target, in particular by parenteral routes.

[0011] For example, severe chronic pain is known to be treated with ziconotide, which is a synthetic equivalent of T co-conotoxin MVIIA, isolated from Conus magus.

[0012] Ziconotide is a peptide that inhibits N-type Cavs found on neurons. Ziconotide prevents calcium from entering nerve endings, which then stop them from releasing their mediators.

[0013] Ziconotide is marketed as PRIALT® as a centrally acting analgesic drug. When delivered to the spinal cord and into the dorsal horns, ziconotide blocks presynaptic Cav2.2 and prevents the entry of calcium in response to action potentials traveling through nociceptive afferent fibers, and the release of neurotransmitters and neuropeptides into the synapse. Therefore, there is no stimulation of postsynaptic neurons, which prevents transmission of the pain signal to the brain. Under the action of ziconotide, the transmission of the pain message at the synaptic level in the dorsal horns of the spinal cord is reduced.

[0014] Inactivated by enzymatic hydrolysis via peptidases, and unable to cross the blood-brain barrier, ziconotide has been granted marketing authorization to treat severe and refractory chronic pain, resistant to opioids. However, ziconotide is authorized only for intraspinal administration in solution, i.e., administration by infusion through a catheter of a ziconotide solution directly between the spinal cord and the dura mater, which allows rapid mixing and diffusion into the cerebrospinal fluid. Intraspinal administration of ziconotide generates an analgesic action.

[0015] Via intraspinal administration, ziconotide acts permanently, as close as possible to the Cav2.2 target at the level of the posterior horn of the spinal cord, where it will then inhibit the release of mediators such as substance P or glutamate, thus reducing pain.

[0016] However, taking into account its pharmaceutical form and its intraspinal administration method, ziconotide PRIALT® remains a limited prescription, a last resort for the treatment of severe chronic pain and reserved for patients for whom even intrathecal morphine does not provide relief.

[0017] Indeed, ziconotide in solution, administered intraspinally, presents several risks of adverse effects.

[0018] Following its intraspinal administration, ziconotide can cause central side effects, such as neuropsychiatric disorders or confusional states, but also hallucinations, memory loss, panic attacks, anxiety, balance disorders, dysarthria, or even nausea, vomiting, abdominal pain and myalgia, and physiological effects in certain patients such as an asymptomatic elevation of Creatine Phospho Kinase.

[0019] In addition to these central, cognitive, and physiological disorders, intraspinal administration is an invasive route of administration for the organism being treated. Indeed, the intraspinal route presents an infectious risk in itself, particularly through the placement of the catheter itself, or associated with the implantable or external pump.

[0020] Furthermore, in addition to the risk of infection, intraspinal administration requires the intervention of a healthcare professional and does not allow the patient to manage their pain by controlling the dosage of the ziconotide solution.

[0021] Consequently, in order to avoid the use of opiates and with the aim of treating pain with peptides of the co-conotoxin type, while limiting the side effects and the risks of infection, the present invention aims to overcome the drawbacks of the state of the art by proposing a pharmaceutical painkiller composition for topical use and action, comprising as active ingredient, one or more peptide(s) of the co-conotoxin family, of natural or synthetic origin, or one of their variant(s), natural or synthetic^), functionally active, for use in a method of treating acute or chronic pain, of a nociceptive, neuro- thic or nociplastic pain, such as intense chronic inflammatory and neuropathic pain. Specifically to the invention, said composition is administered locally, via the skin, near and at the level of the free cutaneous endings of the nociceptors innervating the skin.

[0022] Preferably, the composition of the invention comprises a single type of peptide from the co-conotoxin family, preferably of synthetic origin.

[0023] According to the invention, pain is classified according to its duration over time.

[0024] "Acute pain" is understood to be a warning symptom, transient in time, which corresponds to an unpleasant physiological sensory response linked to the stimulation of peripheral nociceptors by mechanical, thermal, or chemical stimuli.

[0025] In contrast, "chronic pain" is pain that lasts over time, at least beyond 3 months, and persists even if the cause of the pain has disappeared. In the case of chronic pain, the sensation of pain is no longer a warning signal but becomes a disease in its own right with physical, moral and social consequences.

[0026] Additionally, pain is also classified according to its nature.

[0027] In the present invention, a distinction is made between “nociceptive pain”, “neuropathic pain” and “nociplastic pain”.

[0028] "Nociceptive pain" is pain related to the stimulation of nociceptors by a chemical (e.g., inflammation), thermal (e.g., heat source generating a burn), or mechanical (e.g., crushing / cut) stimulus. Nociceptive pain is a response to an attack on the body or its dysfunction in response to trauma, injury, or infection that damages tissues or organs and creates an abnormality. Nociceptive pain generally disappears quickly after the cause of the pain is removed.

[0029] "Neuropathic pain" is caused by an injury or dysfunction of the central or peripheral nervous system. This injury or dysfunction follows, for example, trauma (section or crushing of a nerve, etc.), hypoxia (stroke, compression of a nerve, etc.), a metabolic disease (diabetes), a neurodegenerative pathology, an infection (HIV) or exposure to a toxic agent (cancer chemotherapy).

[0030] “Nociplastic pain” results from an alteration of nociception, without the origin of the alteration being obvious (lesion, inflammation), without stimulation of peripheral nociceptors, or characterized nervous damage.

[0031] For example, the composition of the invention is suitable for use in a method of treating acute nociceptive pain such as post-traumatic pain (due to shock, cut, burn, fracture, etc.), pain resulting from a medical procedure (post-surgical, dressing change, suture, catheter placement, etc.), or pain linked to inflammation of a tissue or organ, for example in response to a metabolic dysfunction (gout), a tumor, an infection by a pathogen or the presence of a foreign body.

[0032] Furthermore, the composition of the invention is also suitable for use in a method of treating chronic nociceptive pain such as low back pain, rheumatoid arthritis, osteoarthritis, chronic primary orofacial pain, autonomic trigerminal headaches, chronic musculoskeletal pain, chronic post-operative pain (such as after amputation, thoracotomy, spinal surgery, etc.) or chronic post-traumatic pain (such as burns and musculoskeletal injuries, etc.).

[0033] For example, the composition of the invention is also suitable for use in a method of treating chronic peripheral neuropathic pain in indications such as cancer, iatrogenic neuropathies induced by chemotherapy, post-radiation or linked to chronic pathologies such as diabetes or AIDS. Among chronic neuropathic pain, there are for example surgical scars, peripheral sequelae pain, radiculopathies, trigerminal or post-shingles neuralgia, lesions of a peripheral nerve, polyneuropathy, neurodegeneration, multiple sclerosis, complex regional pain syndrome type I (soft tissue or bone lesions) or type II (nerve lesion) and mechanical or thermal allodynia, hyperpathia.

[0034] For example, the composition of the invention is also suitable for treating pruritus.

[0035] According to another example, the composition of the invention is also suitable for use in a method of treating chronic nociplastic pain such as fibromyalgia, endometriosis and algodystrophy.

[0036] Preferably, the pharmaceutical composition of the invention is for use in a method of treating inflammatory pain, post-surgical pain or neuropathic pain.

[0037] According to the invention, "topical use and action" means that the pharmaceutical composition is for local, non-systemic use and whose active peptide ingredient of co-conotoxin is not intended to pass into the blood system.

[0038] "By cutaneous route" is understood to mean that the composition is administered by cutaneous route, at the skin level, with a view to a local action of its active peptide ingredient of co-conotoxin, as opposed to a route of administration involving a general, systemic action, with passage into the san- guinea pig. For the purposes of the invention, the term "cutaneous route" excludes transdermal or percutaneous routes. The term "cutaneous route" also does not imply parenteral routes of administration requiring, for example, the use of a needle injection through the skin and therefore excludes transcutaneous routes of administration of the subcutaneous type.

[0039] The term "nociceptors innervating the skin" refers to sensory receptors innervating the skin. Nociceptors are in the form of nerve fibers called "sensory fibers" with a free peripheral ending and a spinal ending. Nociceptors correspond in particular to the free nerve endings of the primary afferent nerves, which are capable of acquiring and then transmitting the stimuli generating painful sensitivity. These free endings are sensory receptors sensitive to mechanical and / or chemical and / or thermal stimuli which can be located in particular in the epidermis, the dermis and the hypodermis; we then speak of "free" cutaneous "ending" or "fiber" of the nociceptors innervating the skin.

[0040] The nociceptors innervating the skin are in direct contact with the environment and allow, following stimulation, the creation and then transmission of an electrochemical signal called a “nerve impulse” or “action potential”.

[0041] Nociceptors can be of three types: A[3, Aô “fibers” and C fibers.

[0042] The Aô fibers are myelinated and conduct the action potential rapidly (at an average speed of 20 m / s). The initial message carried by the Aô fibers corresponds to the first stage of the pain felt, which is an acute intense pain that fades quickly.

[0043] C fibers are unmyelinated and conduct the action potential more slowly than Aδ fibers. The initial message carried by C fibers corresponds to the second stage of pain, which is a more diffuse, less intense, less localized pain that lasts longer.

[0044] A|3 fibers are moderately myelinated and conduct the action potential at an average speed of 30 to 120 m / s.

[0045] The local administration, by cutaneous route, of the pharmaceutical composition of the invention comprising one or more peptide(s) of the co-conotoxin family is a non-invasive solution. Consequently, the composition of the invention advantageously makes it possible to overcome the infectious risks linked to an invasive parenteral route, in particular with a catheter, of the intraspinal or subcutaneous administration type.

[0046] It has been unexpectedly found that local administration, by cutaneous route, of the composition of the invention comprising as active principle a coconotoxin, for example MVIIA, near and at the level of the terminals free peripheral cutaneous nociceptors, makes it possible to eliminate the secondary risks observed with parenteral administration, in particular intraspinal administration, while retaining the known pain-relieving effect linked to an action on the Cav.

[0047] Furthermore, according to other characteristics of the invention, the pain-relieving pharmaceutical composition of the invention has a peripheral analgesic effect involving a local action mechanism, of said peptide(s), at the level of the free cutaneous endings A[3 and / or Aô and / or C of the nociceptors innervating the skin.

[0048] The term "cutaneous nociceptor" refers to all types of nociceptors, innervating the skin, responding to one or more stimuli of mechanical, thermal^) or chemical origin by the transmission of a nervous message perceived as a sensation of pain by the body.

[0049] Examples of "cutaneous nociceptors" innervating the skin include mechano-nociceptors, thermo-nociceptors and polymodal nociceptors.

[0050] Mechano-nociceptors respond and are sensitive to mechanical stimuli such as deformation, pressure, rupture, cutting or incision in the skin.

[0051] “Thermal nociceptors” respond and are sensitive to a variation in hot or cold temperature, perceived at the skin level.

[0052] “Polymodal nociceptors” respond and are sensitive to several mechanical, thermal or chemical stimuli.

[0053] Advantageously, the composition of the invention makes it possible to treat pain by a peripheral analgesic action of the co-conotoxin peptide(s), at the level of the free cutaneous endings of the A[3 and / or Aô and / or C fibers.

[0054] Indeed, the analgesic effect on the sensation of pain has been observed by simple administration, by cutaneous route, of the composition of the invention, comprising one or more co-conotoxin peptide(s). Thus, the analgesic effect obtained in the presence of the co-conotoxin peptide does not require its diffusion into the blood and a systemic action.

[0055] According to another characteristic of the pain-relieving pharmaceutical composition of the invention, said peptide(s) physiologically target(s) the free endings of cutaneous nociceptors impacting the function of the voltage-dependent calcium channels called "Cav" involved in the generation and transmission of the nervous message at the origin of the sensation of pain, preferably N-type Cav voltage-dependent calcium channels, more preferably Cav2.2 voltage-dependent calcium channels.

[0056] According to one embodiment of the pain-relieving pharmaceutical composition of of the invention, said active principle essentially comprises one or more peptide(s) selected from the following list of peptides: MVIIA of SEQ ID NO 1, MVIIB of SEQ ID NO 2, MVIIC of SEQ ID NO 3, MVIID of SEQ ID NO 4, GVIA of SEQ ID NO 5, GVIID of SEQ ID NO 4, GVIA of SEQ ID NO 5, GVIIA of SEQ ID NO. NO 6, GVIIB of SEQ ID NO 7, SVIA of SEQ ID NO 8, SVIA mutl of SEQ ID NO 9, SVIA mut2 of SEQ ID NO 10, SVIB of SEQ ID NO 11, SO3 of SEQ ID NO 12, CnVIIA of SEQ ID NO 13, CnVIIB of SEQ ID NO 14, CnVIIA of SEQ ID NO 15, CnVIA of SEQ ID NO 15 CnVIIH of SEQ ID NO 16, Bu8 of SEQ ID NO 17, Cl 16a of SEQ ID NO 18, MoVIB of SEQ ID NO 19, MoVIB of SEQ ID NO 20, PnVIA of SEQ ID NO 21, PnVIB of SEQ ID NO 22, RsXXVIA of SEQ ID NO 24, RsXVIA of SEQ ID NO 24, T VIA CVIA of SEQ ID NO 25, CVIB of SEQ ID NO 26, CVIC of SEQ ID NO 27, CVID of SEQ ID NO 28, CVIE of SEQ ID NO 29, CVIF of SEQ ID NO 30, RVIA of SEQ ID NO 31 and FVIA of SEQ ID NO 32 , or natural variant(s) thereof, or synthetic(s),functionally active(s) exhibiting between 80% and 99% sequence homology.,

[0057] Selon un mode de réalisation privilégié de la composition pharmaceutique de l'invention, ledit principe actif est constitué d'un ou plusieurs peptide(s) choisi(s) parmi la liste suivante de peptides : MVIIA de SEQ ID NO 1, MVIIB de SEQ ID NO 2, MVIIC de SEQ ID NO 3, MVIID de SEQ ID NO 4, GVIA de SEQ ID NO 5, GVIIA (SNX -178) de SEQ ID NO 6, GVIIB de SEQ ID NO 7, SVIA de SEQ ID NO 8, SVIA mutl de SEQ ID NO 9, SVIA mut2 de SEQ ID NO 10, SVIB de SEQ ID NO 11, SO3 de SEQ ID NO 12, CnVIIA de SEQ ID NO 13, CnVIIB de SEQ ID NO 14, CnVIIC de SEQ ID NO 15, CnVIIH de SEQ ID NO 16, Bu8 de SEQ ID NO 17, Cl 16a de SEQ ID NO 18, Mo VIA de SEQ ID NO 19, MoVIB de SEQ ID NO 20, PnVIA de SEQ ID NO 21, PnVIB de SEQ ID NO 22, RsXXVIA de SEQ ID NO 23, T VIA de SEQ ID NO 24, CVIA de SEQ ID NO 25, CVIB de SEQ ID NO 26, CVIC de SEQ ID NO 27, CVID de SEQ ID NO 28, CVIE de SEQ ID NO 29, CVIF de SEQ ID NO 30, RVIA de SEQ ID NO 31 et FVIA de SEQ ID NO 32, or, one of their variants),natural or synthetic, functionally active, exhibiting between 80% and 99% sequence homology.,

[0058] Table 1 below lists the aforementioned peptide sequences, which are either of natural or synthetic origin.

[0059] Table 1

[0060] According to a particular example of the invention, the co-conotoxin peptide(s) specifically target(s) Cav 2.2 and correspond to the peptide MVIIA of SEQ ID NO 1 and / or GVIA of SEQ ID NO 5 and / or CVID of SEQ ID NO 28 taken alone, or, in a mixture.

[0061] For the purposes of the invention, the term "peptide" designates a chain of amino acids linked together by a peptide bond or amide bond. The term "amino acids" designates all amino acids known to those skilled in the art, both in their levorotatory (L) and dextrorotatory D forms, preferably in their natural L form.

[0062] The standard 1-letter amino acid code is used in the figures to define the peptide sequence listing, as defined by Table 2 below.

[0063] Table 2:

[0064] A "natural or synthetic variant" means an amino acid sequence, whether naturally occurring or synthetic, having one or more amino acids modified with respect to one of SEQ ID NOS 1 to SEQ ID NOS 31. The variant may have "conservative" modifications, in which a substituted amino acid has similar structural or chemical properties, such as, for example, the replacement of leucine with isoleucine. A variant may also have "non-conservative" modifications, such as, for example, the replacement of a glycine with a tryptophan. Analogous minor variations may also include additions or deletions of amino acids, or both.

[0065] The term "functionally active variant" means a peptide sequence which retains the biological pain-relieving activity involving a local action mechanism, at the level of the free cutaneous A[3 and / or Aô and / or C terminals of the nociceptors innervating the skin, i.e. a peptide variant whose peripheral analgesic effect is retained. A person skilled in the art is able to determine, using known computer programs or other methods of the state of the art, which amino acid residues can be substituted, inserted, deleted or modified without abolishing the analgesic effect observed following the administration, by the cutaneous route, of the composition of the invention comprising the peptide or its variant.The biological activity and the analgesic effect of the peptide, or its variant, can be tested by a person skilled in the art using known techniques for assessing the pain tolerance threshold such as, for example, the Hargreaves test (Menghon et al, 2017, DOI: 10.21769 / Bio-Protoc.2506) or the Von Frey test (Matthew JG Bradman et al, 2015, DOI: 10.1016 / j; jneumeth.2015.08.010).

[0066] The term "sequence identity" or "homology" refers to the sequence identity or sequence similarity between two polypeptides. When a position in both peptide sequences being compared is occupied by the same amino acid monomer, then the peptides are homologous or identical at that position. The percentage of homology / identity between two sequences is a function of the number of matching positions shared by the two peptide sequences divided by the number of positions being compared multiplied by 100. For example, if 6 of the 10 positions in two sequences match then the two sequences are 60% identical. Typically, a comparison is performed when two peptide sequences are aligned to give maximum homology / identity.

[0067] According to a first embodiment of the composition of the invention, the peptide or its variant is a peptide of natural origin, extracted from a mollusc of the genus Conus.

[0068] Table 3 below gives for each peptide, of natural origin, the organism from which it is extracted.

[0069] Table 3:

[0070] 0071] It should be noted that all the co-conotoxins in Table 3 have known physiological targets of type N Cavs, more particularly type Cav2.2.

[0072] According to a particular embodiment, the composition of the invention comprises one or more natural peptide(s), comprising or consisting of one or other of SEQ ID NO 1 to SEQ ID NO 31, or one of their natural variant(s), secreted by a mollusc of the genus Conus, as mentioned in the table 1.

[0073] According to another embodiment of the composition of the invention, said peptide(s) or their functionally active variant(s) is / are of synthetic origin.

[0074] Par exemple, les peptides synthétiques suivants, présentant des séquences telle que définie dans le tableau 1, sont susceptibles d'être utilisés dans l'invention : MVIIA de SEQ ID NO 1, MVIIB de SEQ ID NO 2, MVIIC de SEQ ID NO 3, MVIID de SEQ ID NO 4, GVIA de SEQ ID NO 5, GVIIA (SNX -178) de SEQ ID NO 6, GVIIB de SEQ ID NO 7, SVIA de SEQ ID NO 8, SVIA mutl de SEQ ID NO 9, SVIAmut2 de SEQ ID NO 10, SVIB de SEQ ID NO 11, SO3 de SEQ ID NO 12, CnVIIA de SEQ ID NO 13, CnVIIB de SEQ ID NO 14, CnVIIC de SEQ ID NO 15, CnVIIH de SEQ ID NO 16, Bu8 de SEQ ID NO 17, Cl 16a de SEQ ID NO 18, Mo VIA de SEQ ID NO 19, MoVIB de SEQ ID NO 20, PnVIA de SEQ ID NO 21, PnVIB de SEQ ID NO 22, RsXXVIA de SEQ ID NO 23, TVIA de SEQ ID NO 24, CVIA de SEQ ID NO 25, CVIB de SEQ ID NO 26, CVIC de SEQ ID NO 27, CVID de SEQ ID NO 28, CVIE de SEQ ID NO 29, CVIF de SEQ ID NO 30, RVIA de SEQ ID NO 31 and F VIA de SEQ ID NO 32.

[0075] As an illustration and non-limiting example of the invention, Table 4 gives examples of peptide variants, of synthetic origin, which are likely to be used in the composition of the invention.

[0076] Table 4:

[0077] active ingredient is a co-conotoxin MVIIA of sequence SEQ ID NO 1; or; one of its natural or synthetic, functionally active variants having between 80% and 99% sequence homology with SEQ ID NO 1.

[0079] Indeed, it has been shown that co-conotoxin, in particular of the synthetic MVIIA type, administered via the skin using a pharmaceutical composition of the invention, causes the blocking of Cav2.2 channels by acting locally at the level of the endings of cutaneous sensory fibers and by reducing neuronal hyperexcitability in an inflammatory and physiological context.

[0080] Thus, local administration, via the skin, of the composition of the invention makes it possible to act on pain sensitivity, and to treat acute or chronic pain, of a nociceptive, neuropathic or nociplastic nature, particularly chronic intense inflammatory or neuropathic chemo-induced pain, by exposing / applying co-conotoxin to peripheral rather than central synaptic terminals than at sensory fiber synapses.

[0081] The composition of the invention, comprising co-conotoxin, is a peripheral analgesic, reducing or even eliminating the sensation of pain by a peripheral action of co-conotoxin at the level of the endings of the cutaneous sensory fibers expressing Cavs, in particular Cav 2.2.

[0082] Advantageously, the composition of the invention does not cause systemic exposure of the co-conotoxin, its diffusion in the bloodstream is limited. This results in a reduction or absence of side effects linked to the administration of the co-conotoxin by intraspinal route. Compared to the intraspinal route, local, cutaneous administration of the co-conotoxin via the composition of the invention did not present any acute toxicity or notable side effects, even at high concentrations of co-conotoxin in animals, as will be shown by the experimental results below.

[0083] The pharmaceutical composition, comprising a co-conotoxin, administered locally via the skin, is an alternative to known treatment methods for severe chronic pain using co-conotoxins, which avoids the problems of toxicity, the risks of infections and the side effects linked to intraspinal administration or other systemic administration.

[0084] According to a preferred embodiment of the invention, said pain-relieving pharmaceutical composition comprises, in mass percentage, relative to its total mass, between 0.0002% and 6% of said co-conotoxin peptide(s), preferably between 0.0004% and 4.1%.

[0085] In these proportions, no acute toxicity was observed in vivo in animals, after cutaneous application of the pain-relieving composition of the invention. In particular, the performance of the mice was evaluated by the horizontal bar test which analyzes the behavior of the mouse and its motor capacity to raise all four legs on a horizontal bar after administration of the treatment. In addition, during the duration of the experiments, the behavior and general condition of the animals was also evaluated by the continuous visual observation test. Nevertheless, a person skilled in the art knows how to test the toxicity of the composition of the invention by any other known methods.

[0086] According to the invention, said pharmaceutical composition is in a pharmaceutically and dermatologically acceptable galenic form, for cutaneous use and topical action.

[0087] For the purposes of the invention, the “galenic form” or “pharmaceutical form” corresponds to the form in which the composition is presented (cream, ointment) which is specially designed for the route of administration for which the composition is intended (cutaneous route and topical action).

[0088] “Pharmaceutically acceptable” means that which is useful in the preparation of the composition and which is safe, stable, non-toxic biologically, nor otherwise undesirable and which is acceptable for human or animal pharmaceutical use.

[0089] "Dermatologically acceptable" means compatibility with the skin, i.e. anything useful in the preparation of the composition that is non-toxic, following cutaneous administration to humans or animals, in particular by topical application. In other words, this corresponds to anything that can be applied to the skin and / or mucous membranes, without risk of causing a reaction such as, for example, an allergic or inflammatory reaction.

[0090] “Cutaneous use” means use for administration by the cutaneous route by application to the skin and / or mucous membranes.

[0091] “Topical action” means an action of the active ingredient of the composition, after direct application, on the skin and / or mucous membranes, preferably on the surface of the skin and / or mucous membranes, which is a local, non-systemic action.

[0092] According to a preferred embodiment, said pain-relieving pharmaceutical composition is compatible with the skin, for direct cutaneous administration.

[0093] The composition of the invention may be in the form of creams, oil-in-water emulsions, water-in-oil or multiple emulsions, solutions, suspensions, or even powders, suitable for application to the skin. The composition of the invention may be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or a mousse. It may also be in solid form, such as a stick, or be applied to the skin in the form of an aerosol.

[0094] Preferably, said pain-relieving pharmaceutical composition of the invention is in a galenic form of paste, ointment, cream, gel, balm, foam, body milk, emulsion, solution, lotion or powder.

[0095] According to another aspect of the invention, the pain-relieving pharmaceutical composition comprises one or more excipients, chosen for example from pharmaceutically and dermatologically acceptable emollients, humectants, antioxidants, emulsifiers, surfactants.

[0096] The term "excipient" means all substances without impact on the physicochemical properties and the therapeutic action of the active ingredient, which are necessary for the establishment of the galenic form of the pain-relieving composition of the invention, not conferring its therapeutic properties but playing a role in its stability and assimilation by the body for local and cutaneous administration.

[0097] According to the invention, the excipients are inert and have no activity towards co-conotoxin. The excipients do not alter in any way the biological action and the peripheral analgesic effect of co-conotoxin, following its cutaneous application via the pain-relieving composition of the invention.

[0098] Of course, the person skilled in the art will take care to choose the nature of the excipients and their proportions, so that the advantageous therapeutic properties of the co-conotoxin or its variant are not, or not significantly, altered by the envisaged addition.

[0099] According to the invention, these excipients can be used alone or in a mixture to formulate the composition of the invention for local administration, via the skin, allowing topical use and action.

[0100] According to a particular embodiment of the invention, the pain-relieving composition is for use in the treatment of channelopathies, such as the treatment of channelopathies involving N-type calcium channels, preferably Cav2.2 N-type calcium channels.

[0101] The term "channel disease" refers to a disease related to a dysfunction or alteration of one or more functions of membrane ion channels.

[0102] Preferably, the pharmaceutical composition of the invention is administered by spreading, via a localized massage action, on the hands and / or feet and / or the painful area of ​​skin to be treated for pain, at least twice a day, preferably morning and evening, preferably with a therapeutically effective amount of between 0.1 mg and 1000 mg, preferably between 3.8 and 396 mg of co-conotoxin peptide(s) / liter of pharmaceutical composition.

[0103] “Therapeutically effective amount” means the amount of peptide(s) sufficient or effective to prevent, treat (stop, prevent progression, inhibit, reduce or slow down) pain and its sensations, including the symptoms and diseases resulting from it.

[0104] According to the invention, the “therapeutically effective quantity” corresponds to the quantity of peptides to be administered to the surface of the skin by massage using the composition of the invention, making it possible to obtain a beneficial effect in the patient, reducing or eliminating their sensation of pain, while respecting the administration dosage.

[0105] The present invention is described in more detail by means of examples and scientific results demonstrating the effect of the composition of the invention. These should not, however, be considered as limiting the scope of the present invention.

[0106] Thus, other characteristics and advantages of the invention will emerge from this detailed description, with reference to the attached figures, in which:

[0107] - [Fig 1] Figure 1 schematically represents an overview of the nerve message transmission pathways involved in the sensation of pain, with in particular the role of Cav2.2 in the spinal synaptic transmission of the pain signal in the absence of treatment (Figure 1 A), after administration of ziconotide by intraspinal route (Figure 1B) or after administration of the composition of the invention by cutaneous route (Figure 1C),

[0108] - [Fig 2] Figure 2 schematically represents a cross-sectional view of the different layers of skin tissue, with the position of the sensory endings of the cutaneous nociceptors,

[0109] - [Fig 3] Figure 3 represents the number of action potentials / second traveling through the mouse saphenous nerve, measured in vitro, on a nerve-skin preparation treated with a solution of MVIIA or lidocaine before the addition of a cocktail of pro-inflammatory compounds,

[0110] - [Fig 4] Figure 4 represents the effect of the administration, intra-plantar or intrathecal, of a solution of MVIIA or morphine, on the pain threshold of mice, evaluated in the Hargreaves test, in an in vivo model of inflammation with carrageenan,

[0111] - [Fig 5] Figure 5 represents the number of action potentials / second traveling through the mouse saphenous nerve as a function of time, measured in vivo in a carrageenan inflammation model in an anesthetized animal, after local application, via the skin, of a cream composition of the invention comprising MVIIA and after thermal stimulation (Figure 5A) (infrared laser) or mechanical stimulation of the skin of the treated area (Figure 5B) (Von Frey test),

[0112] - [Fig 6] Figure 6 represents the effect of local application, by cutaneous route, of a cream composition of the invention comprising MVIIA, on the pain threshold, in an in vivo model of inflammation with carrageenan, evaluated in the Hargreaves test,

[0113] - [Fig 7] Figure 7 represents the effect of local application, by cutaneous route, of a cream composition of the invention comprising MVIIA, on the pain threshold, in an in vivo model of post-surgical pain inflammation, after thermal stimulation in the Hargreaves test (Figure 7A) or after mechanical stimulation in the Von Frey test (Figure 7B),

[0114] - [Fig 8] Figure 8 represents the effect of local application, by cutaneous route, of a cream composition of the invention comprising MVIIA, on the pain threshold in an in vivo model of post-traumatic neuropathic pain, measured by the paw immersion test in cold water (Figure 8A) or in hot water (Figure 8B) or in response to mechanical stimulation in the Von Frey test (Figure 8C).

[0115] As known, as seen in Figure 1 A, the sensation of pain can have different origins. For example, at the thumb 1, a pain message can be induced by a thermal stimulus 2 (e.g., burn), a chemical stimulus 3 (e.g., contact with an acid, a stinging or algogenic substance such as venom; or the release of pro-inflammatory molecules or other chemical mediators by injured tissues or immune cells), or a mechanical stimulus 4 (e.g., pinching of the skin) or an event causing an injury 5, or a dysfunction of a tissue.

[0116] In Figure 1 A, in enlargement 1', at the level of the skin layers 6, the mechanical 4, thermal 2, chemical 3 (example: inflammatory cytokines 91 secreted by immune cells 8) stimulation of the nociceptor receptors will lead to the modulation of the activity of ion channels and a membrane depolarization, or receptor potential 10'. The subsequent regulation of the sodium channels, potassium channels and Cav 7 will generate the action potentials 10 which will propagate to the spinal cord 11, then to the brain 12. They correspond to the nervous message which codes and transports the pain signal.

[0117] In enlargement 11', at synapse 13 between the presynaptic afferent neuron NAP and the postsynaptic neuron NP, the nerve message will be transmitted to the postsynaptic neuron NP present in the spinal cord 11. In response to the electrical signal (action potentials 10), the N-type Cav 7 of the NAP, in particular Cav2.2, open and allow extracellular calcium to enter. This results in an increase in the intracellular calcium concentration in the presynaptic afferent neuron NAP, which induces the fusion of synaptic vesicles 14 with the plasma membrane and the release of excitatory neurotransmitters 15 into synapse 13.These neurotransmitters 15 will act on their channel receptors 71 present on the postsynaptic neuron NP, which will lead to its depolarization and the generation of new action potentials 10 which will be propagated towards the brain 12, where they will be decoded and integrated and will induce the cognitive and emotional aspects of pain.

[0118] In a known manner, as seen in Figure 1B, intraspinal administration 16 of ziconotide 17 into the cerebrospinal fluid, in close proximity to the presynaptic nerve terminal 11' of the primary NAP afferent neuron, blocks the opening of Cav 7 of type Cav2.2, reducing the influx of calcium into the neuron. Consequently, neurotransmitters 15 are not released from vesicles 14 into synapse 13 and the propagation of the nerve signal to the postsynaptic neuron NP is blocked. Thus, intraspinal administration 16 of ziconotide 17 makes it possible to attenuate the perception of pain by blocking the transmission of the nerve signal at synapse 13.

[0119] Surprisingly, as seen in Figure IC, it has been shown that the pharmaceutical composition 18 of the invention comprising a co-conotoxin 19, administered locally, by cutaneous route 20, near and at the level of the free endings of cutaneous nociceptors, exhibits a peripheral analgesic effect involving a local mechanism of action. The peripheral analgesic effect obtained by cutaneous administration 20 of the composition of the invention 18 suggests the involvement of Cav 7 of type Cav 2.2 at the level of the free endings of cutaneous nociceptors.

[0120] More specifically, the present invention has shown that administration, via the cutaneous route, at the level of the endings of cutaneous nociceptors impacts the generation and transmission of the nerve pain message, even though the co-conotoxin is not present near the spinal presynaptic nerve ending.

[0121] The present invention has shown that the administration of co-conotoxin by intraspinal route directly at the presynaptic terminal is not necessary to obtain an analgesic effect. Indeed, a local, non-systemic peripheral action of co-conotoxin, administered by cutaneous route, also makes it possible to limit the transmission of the nerve pain message as confirmed by the experimental results below.

[0122] As seen in Figure 2, the sensory fiber endings of cutaneous nociceptors, for example of the Aδ fiber or C fiber or A|3 fiber type, have projections into the dermis and epidermis. The present invention shows the role of these projections, in the presence of co-conotoxin, in a nociceptive pathway involving Cavs and the emission of a pain signal.

[0123] Advantageously, the results of experiments below have shown that the local administration, via the skin, of the composition of the invention comprising a co-conotoxin peptide, impacts the propagation of the pain message, this by simple administration near the free endings of the cutaneous nociceptors.

[0124] In other words, free nerve endings and projections of cutaneous nociceptors at the level of the epidermis and dermis are sensitive to co-conotoxin peptides, when they are applied locally using the composition of the invention via the cutaneous route.

[0125] Example of a pharmaceutical painkiller composition for local administration, via the skin.

[0126] Lipophilic emulsion:

[0127] Water, Liquid Paraffin, Paraffin, Sorbitan Isostearate, Petrolatum, Sorbitan Laurate, Hydrogenated Castor Oil, PEG-25, Beeswax, Hydrogenated Castor Oil, Polysorbate 20, Benzyl Alcohol, Magnesium Sulfate, Phenoxyethanol, Stearic Acid, Potassium Sorbate, Fragrance, Citric Acid, Alpha-Isomethyl Ionone, Alpha-Amylcinnamic Aldehyde, Benzyl Benzoate, Benzyl Salicylate, Limonene, which is a lipophilic emulsion, Water in occlusive oil with emollient power, with a lipid content of 54% and water of 46% marketed under the name Excipial lipocrème CR T / 50G and forming the cream support base, to which synthetic MVIIA is added in solution at different concentrations.

[0128] The above composition is given as an example, without however limiting the scope of protection of the invention.

[0129] The experimental results below are intended to demonstrate the peripheral analgesic effect obtained by application to the skin of a composition of the invention, in particular by implementing the scientific tests and observations described below.

[0130] Principle of the Hargreaves test (thermal stimulation):

[0131] Thermal stimulation by infrared radiation consists of the Hargreaves plantar test which allows the evaluation of the thermal pain threshold. This test allows the measurement of the nociceptive threshold of an animal, of the rodent type, whose plantar surface is subjected to an infrared thermal stimulus. The animals are placed in a transparent cage. An infrared beam is used to locally stimulate the paw, in particular the inflamed paw. The pain threshold in response to this thermal stimulation is evaluated by measuring the paw withdrawal time or "withdrawal latency". A longer withdrawal latency after treatment means an increase in the nociceptive threshold and therefore an increased tolerance to pain. In the present invention, the implementation of the Hargreaves test is carried out in accordance with the protocol described in the publication by Menghon et al, 2017, DOI: 10.21769 / BioProtoc.2506, the contents of which must be considered as part of this application.

[0132] Principle of the Von Frey test (mechanical stimulation):

[0133] The Von Frey tactile sensitivity test (with a method for calculating Chaplan's tactile sensitivity described in the publication SR Chaplan et al, 1994, Journal of Neuroscience Methods, DOI: doi.org / 10.1016 / 0165-0270(94)90144-9) consists of individually placing rodent-type animals in cages with a wire mesh floor, then applying a painful model perpendicular to the paw, for example on a paw sensitized by inflammation, mechanical pressure by calibrated monofilaments of different mechanical forces. Any reaction of the animal such as withdrawal of the paw, licking or vocalization is considered a positive response, following perceived pain. In the present invention, the implementation of the Von Frey test is carried out in accordance with the protocol described in the publication of Matthew JG Bradman et al, 2015, DOI: 10.1016 / j; jneu-meth.2015.08.010, the contents of which should be considered as part of this request.

[0134] Principle of the horizontal bar test (motor coordination):

[0135] This test consists of presenting a raised horizontal bar to an animal, such as a rodent, held by the tail so that it grasps it with its forelimbs, then measuring the time required for the animal to recover with its four presses on this bar. A decrease in performance (increase in the time required to recover) will suggest a potential adverse effect of the treatment on one or more physiological or behavioral functions such as motor coordination, muscle strength, motivation or vigilance of the animal.

[0136] Principle of the visual observation test (side effect on behavior):

[0137] This test involves regularly observing the general behavior of mice during in vivo experiments to identify unexpected and significant effects resulting from the treatment or procedure being performed. For example, it is observed whether the animal maintains normal movement or reaction activity or exhibits abnormal behavior. This test is performed by a qualified experimenter who is familiar with the normal behaviors and activity expected in the animal. For example, behavior close to sedation or prostration, excessive grooming, random exploration or movement, piloerection, scratching, vocalizations, tail straightness / rigidity may be observed.

[0138] These tests were implemented in the experiments below. The results obtained show the effect of local administration, by cutaneous route, near the endings of the fibers of the cutaneous nociceptors of a pharmaceutical composition comprising MVIIA in a method of treatment of intense chronic inflammatory and neuropathic pain.

[0139] A) Demonstration of the effect of an MVIIA solution on the activity of peripheral sensory fibers in response to inflammatory chemical stimulation, in an ex vivo electrophysiological peripheral model (Figure 3)

[0140] The study aims to record the action potentials running through the saphenous nerve, on an ex vivo model of a dissected paw, after chemical stimulation of the inner surface of the skin by an "inflammatory cocktail", in the presence or absence of MVIIA or lidocaine.

[0141] Experiments were performed on commercial male C57B1 / 6J mice (Janvier Labs) aged 5-8 weeks, with a total weight between 20-25g.

[0142] The measurement of ex vivo nerve action potentials is carried out in a KREBS solution dedicated to this purpose.

[0143] MVIIA co-conotoxin is a solution of commercial MVIIA diluted in a suitable KREBS solution, which is at a concentration of 10 pM.

[0144] Lidocaine is a solution diluted in said KREBS solution at a concentration of 10 mM. Lidocaine serves as a positive control in this experiment. Lidocaine is known as a local anesthetic, an inhibitor of all nerve impulses, acting by blocking voltage-gated sodium channels.

[0145] The inflammatory context is simulated by a cocktail of pro-inflammatory compounds called "inflammatory cocktail" comprising a mixture of bradykinin, prostaglandin E2, serotonin, histamine and potassium chloride. The administration of the inflammatory cocktail stimulates different receptors and generates an increase in the frequency of emission of action potentials in the saphenous nerve.

[0146] After sacrificing the mice, the saphenous nerve was dissected and removed with the skin. Action potential measurement was performed directly ex vivo on the nerve-skin dissection.

[0147] The results obtained are visible in Figure 3.

[0148] Neuronal activity, representative of a nervous message generated in response to inflammatory stimulation, results in the detection of action potentials measured using any device known to those skilled in the art.

[0149] Figure 3 represents the frequency of action potential emission as a function of time, measured in the nerve-skin model, after stimulation by the inflammatory soup, in the presence of MVIIA, lidocaine or the dilution solution (KREBS solution).

[0150] As seen in Figure 3, in this ex vivo peripheral electrophysiological model, treatment with MVIIA solution has an inhibitory effect on the activity of the saphenous nerve stimulated by the inflammatory soup, as does the positive control lidocaine.

[0151] These results confirm that MVIIA has a peripheral neuroinhibitory effect on the endings of cutaneous sensory fibers stimulated by inflammatory or inflammation-mimicking molecules.

[0152] B) Effect of intraplantar or intrathecal administration of a solution of MVIIA or morphine on the pain threshold in an in vivo inflammation model (Figure 4)

[0153] In an in vivo inflammatory model, the study aims to measure the pain threshold in response to thermal stimulation (via the Hargreaves test), after induction of inflammation by carrageenan, and administration of an MVIIA solution either intra-plantarly or intrathecally.

[0154] The objective is to show that peripheral and local intra-plantar injection of MVIIA, in the inflamed paw of an animal, induces a modification of the pain threshold perceived in response to thermal stimulation.

[0155] The experiment implements the aforementioned Hargreaves test.

[0156] This experiment shows the difference in the action of MVIIA on perceived pain, depending on the method of administration. The experiment shows the effect of MVIIA after a local intra-plantar injection in the periphery and the effect of a central intrathecal injection of MVIIA.

[0157] Experiments were performed on commercial CD1 male and female mice (Janvier Labs) with a total weight of 20-25g.

[0158] In this experiment, the induction of inflammation is carried out by injecting a 2% carrageenan solution diluted in physiological serum into the left hind paw of mice in vivo so as to obtain an inflamed paw.

[0159] MVIIA co-conotoxin is a solution of commercial synthetic MVIIA diluted in saline (0.9% NaCl). Morphine is also diluted in saline. The saline corresponds to the vehicle and serves as a negative control.

[0160] Morphine serves as a positive control, this substance being known to inhibit the sensation of pain perceived in response to thermal stimulation.

[0161] For the experiment, the mice were placed individually in the Hargreaves test chambers for its implementation. The pain threshold of the mice was assessed by thermal stimulation of the inflamed paw by the IR beam, before induction of inflammation (T-210), and 3 hours 30 minutes after induction of inflammation (peak inflammation of the paw) and application of the treatment (T0), then at different times after treatment with MVIIA, morphine or the control vehicle. The withdrawal time or latency time was measured every 15 min during the first hour after treatment, then every 30 min, as visible in Figure 4.

[0162] As seen in Figure 4, intraplantar administration of MVIIA leads to an increase in the withdrawal latency of the inflamed paw, in response to thermal stimulation. In particular, 30 min after treatment, the pain sensitivity observed for an intraplantar injection of 1500 pmol of MVIIA is comparable to that observed after the intraplantar injection of 10.5 nmol of morphine.

[0163] The results in Figure 4 show that peripheral local administration of an MVIIA solution induces an analgesic effect in vivo.

[0164] In parallel with the results in Figure 4 allowing the determination of the in vivo analgesic effect of the local application of MVIIA, the effect of the different treatments on the general behavior of the mice was evaluated, throughout the experiment, by implementing the horizontal bar test and the visual observation test mentioned above.

[0165] Under the conditions of the experiment in Figure 4, no major behavioral changes were observed in the animal following treatment with intra-plantar route with MVIIA, or morphine. In contrast, the animal's general behavior was negatively impacted when MVIIA or morphine was administered intrathecally. These results suggest a narrow therapeutic margin for these two treatments using the intrathecal route of administration.

[0166] For the purposes of the invention, the therapeutic margin corresponds to the safety margin of a drug, i.e. the difference between the therapeutic threshold of a drug (minimum concentration below which no activity is obtained) and its toxic threshold (maximum concentration beyond which undesirable effects appear). Drugs with a narrow therapeutic margin are characterized by toxic concentrations close to the effective concentrations.

[0167] During the experiment in Figure 4, the visual observation test and the horizontal bar test were implemented. The results of these tests showed that the performance of animals treated with intraplantar injection of MVIIA did not decrease. No notable behavioral changes were observed in these two tests during the experiment.

[0168] These results suggest better tolerance of treatment by local intraplantar injection of MVIIA than by intrathecal route.

[0169] All these results (Hargreaves test) demonstrate the peripheral analgesic action of intraplantar administration of MVIIA on the sensation of pain caused by thermal stimulation of an inflamed paw. Furthermore, during this experiment, intraplantar application of a therapeutic dose of MVIIA does not have a significant effect on the general behavior of the animal or its performance in the horizontal bar test, contrary to what was observed after administration of MVIIA by intraspinal route, or morphine. These results suggest an increase in the therapeutic margin of MVIIA administered by cutaneous route, at the periphery.

[0170] C) Effect of the administration, by cutaneous route, of a pharmaceutical composition of cream containing MVIIA according to the invention (Figure 5) on nerve signaling in vivo, in anesthetized animals, in models of thermal or mechanical hypersensitivity induced by inflammation

[0171] The study aims to test the effect of peripheral, cutaneous application of MVIIA in the form of a cream, in a carrageenan-induced inflammatory pain model in anesthetized animals. The effect of peripheral application is observed by measuring action potentials traveling along the saphenous nerve in response to mechanical stimulation (Von Frey test) or thermal stimulation (application of an infrared laser).

[0172] In this experiment, in vivo, on anesthetized animal, the behavioral response- response to mechanical stimulation caused by the Von Frey filament is replaced by a recording of action potentials from the entire saphenous nerve on the partially dissected leg.

[0173] As seen in Figure 5A, action potentials traveling along the saphenous nerve in response to nociceptive stimulation of the inflamed area are recorded in vivo in anesthetized animals. Action potentials of the saphenous nerve are recorded following thermal stimulation of approximately 50°C induced by an infrared laser at a wavelength of 980 nm (Figure 5A) and during mechanical stimulation of the skin, innervated by the nerve, with a Von Frey filament calibrated to apply a force of 10.0 g (Figure 5B).

[0174] Experiments were performed on commercial CD1 male and female mice (Janvier Labs) weighing 20-25g.

[0175] In this experiment, the induction of inflammation is carried out by intraplantar injection into a mouse hind paw in vivo of a 2% carrageenan solution, diluted in physiological saline.

[0176] The anesthetic mixture consists of ketamine and xylazine diluted in physiological saline.

[0177] MVIIA is administered cutaneously. MVIIA is presented in the form of a cream consisting of a mixture, volume by volume, of 1 / 3 of a solution of MVIIA diluted in water, with 2 / 3 of a cream carrier base “Excipial lipocrème CR T / 50G” of composition as above for respective final concentrations of MVIIA in the cream of 1.5 pM, 0.15 pM or 0.015 pM. These mixtures represent specific examples of pharmaceutical composition according to the invention.

[0178] After induction of inflammation with intraplantar carrageenan, 100pL of the aforementioned pharmaceutical compositions are administered to the animal by cutaneous massage of the inflamed paw for 60 seconds. Then, the animals are anesthetized and positioned on a platform with demonstration of the saphenous nerve and the saphenous vein by partial dissection of the inflamed paw.

[0179] Recording of nerve action potentials was performed by stimulating the skin with a calibrated 10.0g Von Frey filament or with an infrared laser (wavelength 980nm). The results are shown in Figures 5 A and 5B.

[0180] The results show that in an in vivo inflammatory model, local application, via the skin, of a composition of the invention in the form of a cream, induces a reduction in the frequency of emission of action potentials, at the highest dose, on both types of stimulation, mechanical and thermal, applied to the inflamed skin.

[0181] The decrease in the measured electrical signal (frequency of action potentials) reflects a decrease in the generation and transmission of the nociceptive nerve message which will result in a decrease in the sensation of pain in the animal.

[0182] The application of a composition of the invention comprising MVIIA, in a localized manner, by cutaneous administration, makes it possible to reduce the electrical activity at the origin of the sensation of pain.

[0183] These results suggest the analgesic effect of cutaneous administration of MVIIA, certainly involving local inhibition of Cav2.2 at the peripheral level.

[0184] D) In ​​vivo effect of the cutaneous administration of a pharmaceutical composition of cream containing MVIIA according to the invention in a model of inflammatory pain with thermal stimulation (Hargreaves test) (Figure 6)

[0185] The study aims to measure the analgesic effect induced by the peripheral and local administration, via the skin, of a pharmaceutical composition of the invention comprising MVIIA, on thermal hypersensitivity caused by inflammation, with evaluation of the nociceptive threshold by the aforementioned Hargreaves test.

[0186] Experiments were performed on commercial male and female CD1 mice (Janvier Labs) aged 5 to 8 weeks and weighing 20 to 25g.

[0187] Induction of inflammation is done by intraplantar injection of a 2% carrageenan solution so as to obtain an inflamed paw.

[0188] MVIIA is administered cutaneously. MVIIA is presented in the form of a cream consisting of a mixture, volume by volume, of 1 / 3 of a solution of MVIIA diluted in water, with 2 / 3 of a cream carrier base “Excipial lipocrème CR T / 50G” of composition as above for final concentrations of 1.5pM, 0.15pM or 0.015pM. These mixtures represent specific examples of pharmaceutical composition according to the invention.

[0189] As a first positive control, a lidocaine cream with a final lidocaine concentration of 10 mM was prepared by mixing 1 / 3 water and 2 / 3 “Excipial lipocrème CR T / 50G” cream base, then administered with a volume of 100pL on the inflamed paw by skin massage for approximately 1 minute.

[0190] As 2 ème positive control, morphine, is diluted in physiological serum at a concentration of 26.3 mM for intraperitoneal injection at a dose of 10 pL / g.

[0191] For the experiment, the mice are evaluated in the Hargreaves test device before, then 3h30 after induction of inflammation by intraplantar injection of carrageenan in one of the hind paws. Then, 100pL of the aforementioned MVIIA pharmaceutical compositions and the different te- less are administered to the animal by skin massage for approximately 60 seconds at the level of the inflamed paw or intraperitoneally for morphine. The pain threshold of the mice will then be regularly assessed by thermal stimulation of the inflamed paw with the infrared beam. The paw withdrawal latency is measured every 15 min during the first hour post-treatment, then every 30 min during the remaining time of the experiment, as visible in Figure 6.

[0192] As seen in Figure 6, cutaneous application of MVIIA in cream form significantly increases the nociceptive threshold in mice, with a withdrawal latency of the inflamed paw equivalent to that observed before induction of inflammation, for example at T60 minutes.

[0193] A dose-response effect of MVIIA applied as a cream is also visible, with a maximum effect observed at the intermediate dose of 1.5 nmol of MVIIA. The effects of MVIIA are comparable to those observed with lidocaine (anesthetic) or with a high dose of morphine, while being more sustained over time.

[0194] Dose-response refers to the relationship between the dose and the observed biological effect. Increasing the dose can increase the intensity or severity of an effect. More specifically, the dose-response gives the relationship between the amount of exposure to a substance (dose) and the resulting changes in physiological functions or health (response).

[0195] These results demonstrate the efficacy of MVIIA in cutaneous application on thermal hypersensitivity induced by inflammation.

[0196] Throughout this experiment, the general condition and behavior of the mice was assessed by the visual observation test and by measuring their performance in the horizontal bar test. The results obtained, in parallel with the Hargreaves test in Figure 6, show no deviation in the behavior or general condition of the mice after treatment with MVIIA. No significant effect after treatment with MVIIA, whatever the dose applied, could be observed when performing the horizontal bar test or the visual observation test, unlike treatment with morphine.

[0197] Specifically, in the horizontal bar test, no performance decline was observed in animals treated with MVIIA by cutaneous application, regardless of the dose applied. In particular, no performance decline was observed at the maximum dose tested, 10 times higher than the effective dose of 1.5 nmol. On the other hand, a significant impairment in the performance of mice treated with morphine could be observed in the horizontal bar test. Intraperitoneal morphine impacts, at the therapeutic doses tested, certain central or physiological functions of mice, unlike the tested doses of MVIIA by cutaneous administration.

[0198] The overall results of the experiment in Figure 6 (Hargreaves test, horizontal bar test, visual observation test) show that in an in vivo model of inflammation-induced thermal hypersensitivity, the cutaneous application of an MVIIA cream, at the tested doses (1.5 nmol) and (15 nmol), induces superior analgesia compared to the cream containing lidocaine (1 pmol). This analgesic effect is also comparable to the analgesia obtained by systemic (intraperitoneal) administration of a dose of morphine (7.5 mg / kg), without impact on the general condition and behavior of the animals. These results suggest an improved therapeutic margin compared to intrathecal administration of MVIIA.

[0199] These results confirm the analgesic effect that could be induced by inhibition of Cav2.2 at the peripheral level in an inflammatory model, without inducing significant side effects. The local (topical) application, via the skin, of a pharmaceutical composition of the invention, facilitates the administration of the active ingredient of co-conotoxin, of the MVIIA type, while avoiding the undesirable effects linked to an invasive route of administration of the intrathecal type.

[0200] E) Effect of the administration, by cutaneous route, on an in vivo model of post-surgical pain, of a pharmaceutical composition of cream containing MVIIA according to the invention with thermal and mechanical stimulation (Figures 7A and 7B)

[0201] The study aims to measure the analgesia induced following the administration, at the periphery of the body, by cutaneous route, of MVIIA in the form of a cream in an in vivo inflammatory model of post-surgical pain, with thermal stimulation (Hargreaves test Figure 7A) and mechanical stimulation (Von Frey test Figure 7B).

[0202] Experiments were performed on commercial (Janvier Labs) CD1 male and female mice weighing 20-25g.

[0203] Post-surgical pain is induced by performing on each anesthetized animal (isoflurane) a surgical operation characterized by a subplantar incision and suture according to the known Brennan protocol (Brennan et al, 1996, DOI. 10.1016 / 0304-3959(95)01441-1) the content of which must be considered as part of the present application.

[0204] MVIIA is administered cutaneously. MVIIA is presented in the form of a cream consisting of a mixture, volume by volume, of 1 / 3 of a solution of MVIIA diluted in water, with 2 / 3 of a cream carrier base “Excipial lipocrème CR T / 50G” of composition as above for respective final concentrations of MVIIA in the cream of 0.15 pM or 1.5 pM. These mixtures represent specific examples of pharmaceutical composition according to the invention.

[0205] After manual mixing, 100pL of one of the above-mentioned cream compositions are administered to the animal by cutaneous massage of the paw for 60 seconds, which corresponds to an applied quantity of 1.5 nmol or 0.15 nmol of MVIIA.

[0206] As an experimental control, the cream support base “Excipial lipocrème CR T / 50G”, with 1 / 3 water volume by volume, is applied cutaneously under the same conditions.

[0207] Figure 7A: Measurement of the analgesic effect of MVIIA administered as a cream in a postoperative pain model (thermal modality: Hargreaves test)

[0208] Mice were placed in the Hargreaves test compartments. The baseline threshold was then measured in response to thermal stimulation on the left hind paw.

[0209] After determining the baseline threshold, the mice were operated on according to the Brennan protocol and left to rest for 20 hours.

[0210] After this rest and 1 hour of conditioning in the Hargreaves test cage, the measurement of the nociceptive threshold is carried out at T0 by thermal stimulation.

[0211] Then, the mice were treated by skin massage with 100 μL of cream containing MVIIA, i.e. with an amount of 1.5 nmol or 0.15 nmol of MVIIA, or with the cream base control (vehicle).

[0212] After treatment and administration by cutaneous route, analgesia is assessed every 15 to 30 min by measuring the paw withdrawal latency in seconds under the effect of thermal stimulation.

[0213] The results are visible in Figure 7A and show an increase in the withdrawal latency time following the application of a composition of the invention, by cutaneous route. These results reflect an increase in the pain threshold in animals treated with a composition of the invention administered locally by cutaneous route.

[0214] Figure 7B: Measurement of the analgesic effect of MVIIA, administered in cream form, in a post-operative pain model (mechanical modality: Von Frey test and calculation of tactile sensitivity according to the Chaplan method)

[0215] The mice were subjected to the Von Frey test. The basic threshold of tactile sensitivity was determined by mechanical stimulation of the paw with filaments of increasing strength and calculated according to the Chaplan method (SR Chaplan et al, 1994, Journal of Neuroscience Methods, DOI: doi.org / 10.1016 / 0165-0270(94)90144-9), the content of which should be considered as part of this application.

[0216] After determining the baseline threshold, the mice were operated on according to the Brennan protocol and left to rest for 20 hours.

[0217] According to the Von Frey test, at time zero T0, the evaluation of the nociceptive threshold is carried out, by mechanical stimulation of the operated area, with filaments calibrated in accordance with the Von Frey test.

[0218] Then, the mice were treated by cutaneous massage of 100 μl of cream containing an amount of 1.5 nmol or 0.15 nmol of MVIIA or with the cream base control. After treatment and cutaneous administration, analgesia was evaluated every 30 min, by calibrated monofilaments in the Von Frey test and the sensitivity threshold calculated according to the Chaplan method.

[0219] The results presented in Figure 7B show an increase in withdrawal latency following the application of a composition of the invention by the cutaneous route.

[0220] In the experiments in Figure 7A and 7B, assessing post-surgical thermal and mechanical sensitivity after cutaneous treatment with MVIIA, the general condition and behavior of the mice were monitored by the horizontal bar test and the visual observation test. In the context of these experiments, no deviations in the general condition and behavior of the treated animals were reported.

[0221] This study, which aimed to test MVIIA in the form of a pharmaceutical composition administered locally via the skin, on an in vivo model of post-operative pain with two stimulation modalities (thermal and mechanical), shows that this local administration method, via the skin, effectively induces an analgesic effect.

[0222] The pharmaceutical composition of the invention therefore induces an analgesic effect on post-surgical pain following local peripheral administration, via the skin.

[0223] F) Effect of administration, by cutaneous route, on an in vivo model of traumatic neuropathic pain, of a pharmaceutical composition of cream containing MVIIA according to the invention, and evaluation of thermal hypersensitivity with thermal stimulation of the paw immersion at 10°C and 46°C (Figures 8A and 8B), or of mechanical hypersensitivity (Von Frey test (Figure 8C)) resulting.

[0224] The study aims to measure the analgesia induced following peripheral administration, by the cutaneous route, of MVIIA in the form of a cream, in the in vivo model of chronic neuropathic pain induced by the spared nerve injury model, inducing allodynia (hypersensitivity) to thermal and mechanical stimulation.

[0225] Post-surgical neuropathic pain is induced by performing on each anesthetized animal a surgical operation to section the peroneal and tibial branches of the sciatic nerve as indicated in the publication by Anne Frédérique Bourquin et al, 2006, DOI: 10.1016 / j.pain.2005.10.036, the content is part of this application.

[0226] This operation induces in a few days a mechanical and thermal hypersensitivity (allodynia) of the area innervated by the 3rd branch or sural nerve.

[0227] Experiments were performed on commercial (Janvier Labs) CD1 male and female mice weighing 20-25g.

[0228] Ligation and section were performed under anesthesia (isoflurane) and the animals were left to recover with observation by the regular visual observation test. Evaluation of thermal or mechanical hypersensitivity was performed 14 days after induction of the model.

[0229] MVIIA is administered cutaneously. MVIIA is presented in the form of a cream consisting of a mixture, volume by volume, of 1 / 3 of a solution of MVIIA diluted in water, with 2 / 3 of a cream carrier base “Excipial lipocrème CR T / 50G” of composition as above for respective final concentrations of MVIIA in the cream at 1.5 pM or 0.15 pM. These mixtures represent specific examples of pharmaceutical composition according to the invention.

[0230] After mixing, 100pL of the cream is administered to the animal by cutaneous massage of the paw for 60 seconds, i.e. to administer a quantity of 1.5 nmol or 0.15 nmol of MVIIA

[0231] As an experimental control, the cream support base “Excipial lipocrème CR T / 50G”, added volume to volume of 1 / 3 water, called “vehicle” is applied cutaneously.

[0232] Figure 8A and 8B: Measurement of the analgesic effect on neuropathic pain in a model of induced hot and cold thermal allodynia (hypersensitivity) (10°C or 46°C) (nerve-spared model)

[0233] A mouse hind paw is immersed in cold (10°C) or warm (46°C) water. Then the baseline threshold of the time taken to withdraw the immersed paw from the water is measured.

[0234] After determining the baseline threshold, the mice were operated on to sever the peroneal and tibial branches of the sciatic nerve. After the operation, the mice were left to rest for 14 days, where postoperative monitoring using the visual observation test was performed.

[0235] After this rest, the evaluation of thermal sensitivity is carried out (time T0) by measuring the withdrawal latency of the paw immersed in water at 10°C or 46°C.

[0236] Then, the mice are treated by skin massage with 100 μL of one of the creams of the invention either with a dose of 0.15 nmol or 1.5 nmol of MVIIA, in parallel with a control cream devoid of MVIIA.

[0237] After treatment and administration by cutaneous route, the evaluation of the induced analgesia is carried out every 30 min, by measuring the withdrawal latency of the paw in seconds under the effect of thermal stimulation by immersion in cold water at 10°C or hot water at 46°C.

[0238] The results are seen in Figure 8A (immersion in water at 10°C) and Figure 8B (immersion in water at 46°C).

[0239] These results show an increase in withdrawal latency time in response to the application of a pharmaceutical composition of the invention by cutaneous route on induced post-operative neuropathic pain.

[0240] Figure 8C: Measurement of the analgesic effect via mechanical stimulation (Von Frey test) in the context of post-operative neuropathic pain

[0241] The Von Frey test is performed on mice.

[0242] Then the baseline threshold measurement is performed. The baseline threshold corresponds to the measurement of the mechanical pain threshold, calculated according to the Chaplan method, involving the evaluation of paw withdrawal, in response to the application of Von Frey filaments of increasing forces.

[0243] After determining the baseline threshold, the mice were operated on to sever the peroneal and tibial branches of the sciatic nerve according to the protocol of the aforementioned publication by Anne Frédérique Bourquin et al, 2006. After the operation, the mice were left to rest for 14 days where postoperative monitoring was carried out.

[0244] After this rest, the evaluation of tactile sensitivity at time zero T0 is carried out after mechanical stimulation by Von Frey filaments of increasing strength and according to the Chaplan calculation method.

[0245] Then, the mice are treated by skin massage with 100 μL of one of the creams of the invention either with a dose of 0.15 nmol or 1.5 nmol of MVIIA or with the cream base control (vehicle).

[0246] After treatment and cutaneous administration, analgesia is assessed every 30 min, by assessing the force inducing withdrawal of the paw subjected to mechanical stimulation by Von Frey filaments of increasing strength.

[0247] The results are seen in Figure 8C.

[0248] These results show an increase in pain tolerance following the application of a composition of the invention, via the skin, in a model of mechanical allodynia induced by post-traumatic neuropathy.

[0249] Throughout the experiment, the general condition and behavior of the animals were regularly observed using the aforementioned visual observation test. No notable deviant or unusual behavior was detected in these experiments assessing the general condition of the animals, either in the MVIIA-treated animals or in the control.

[0250] All the results in Figure 8 show that the local administration, by cutaneous route, of a composition of the invention comprising MVIIA, in an in vivo model of post-traumatic neuropathic pain, generates an analgesic effect on pain caused by immersion in cold or hot water, or by mechanical stimulation of the hypersensitive area.

[0251] Study of the bioavailability of MVIIA in plasma, following the application of a composition of the invention, by cutaneous route:

[0252] The concentration of MVIIA in the plasma of mice was studied after local cutaneous application of the compositions of the invention used in the above-mentioned experiments.

[0253] The concentration of MVIIA is measured by quantification of MVIIA in the plasma of treated mice, as a function of time, by mass spectrometry according to any methods known to those skilled in the art.

[0254] The results obtained show that MVIIA is not detected in plasma samples taken from treated mice, with a detection threshold of around 10 nM.

[0255] Thus, the analgesic and pain-relieving effect after application, by the cutaneous route, of a composition of the invention, is obtained without the need for MVIIA to be present in the plasma and without systemic exposure.

[0256] Thus, all of these experimental results show that the local application, via the skin, of a pain-relieving pharmaceutical composition comprising a co-conotoxin, in particular of the MVIIA type, in the form of a cream, intended for topical application to the skin, makes it possible to relieve intense chronic inflammatory and neuropathic pain, by a peripheral analgesic effect via the sensory endings of cutaneous nociceptors, in particular involving Cavs, in particular of the Cav2.2 type.

[0257] The present pain-relieving pharmaceutical composition, administered locally via the skin, therefore constitutes an alternative solution to the intraspinal route of treatment of intense chronic pain by a co-conotoxin and to the use of opioids in general.

[0258] This new method of administering co-conotoxin, involving a peripheral analgesic effect, advantageously allows us to avoid the undesirable effects and infectious risks linked to treatment using the intraspinal route, while allowing direct pain management by the patient who is able to easily administer the composition by skin massage.

Claims

Claims

1. Pain-relieving pharmaceutical composition for topical use and action, comprising as active ingredient, one or more peptide(s) of the co-conotoxin family, of natural or synthetic origin, or one of their variant(s), natural or synthetic, functionally active, for use in a method of treating acute or chronic pain, of a nociceptive, neuropathic or nociplastic nature, such as intense chronic inflammatory and neuropathic pain, characterized in that it is administered locally, via the skin, close to and at the level of the free cutaneous endings of the nociceptors innervating the skin.

2. Pain-relieving pharmaceutical composition, according to the preceding claim, characterized in that it has a peripheral analgesic effect involving a local action mechanism, of said peptide(s), at the level of the free cutaneous endings A[3 and / or Aô and / or C of the nociceptors innervating the skin.

3. Pain-relieving pharmaceutical composition according to any one of the preceding claims, characterized in that said peptide(s) physiologically target(s) the free endings of cutaneous nociceptors impacting the function of the voltage-dependent calcium channels known as "Cav" involved in the generation and transmission of the nerve message causing the sensation of pain, preferably N-type Cav voltage-dependent calcium channels, more preferably Cav2.2 voltage-dependent calcium channels.

4. Pharmaceutical anti-pain composition, according to one of the previous claims, characterized by the fact that the main action is made up of one or more peptide(s) chosen from the following list of peptides: MVIIA of SEQ ID NO 1, MVIIB of SEQ ID NO 2, MVIIC of SEQ ID NO 3, MVIID de SEQ ID NO 4, GVIA de SEQ ID NO 5, GVIIA (SNX -178) de SEQ ID NO 6, GVIIB de SEQ ID NO 7, SVIA de SEQ ID NO 8, SVIAmutl de SEQ ID NO 9, S VIA mut2 de SEQ ID NO 10, SVIB de SEQ ID NO 11, SO3 de SEQ ID NO 12, CnVIIA de SEQ ID NO 13, CnVIIB de SEQ ID NO 14, CnVIIC de SEQ ID NO 15, CnVIIH de SEQ ID NO 16, Bu8 de SEQ ID NO 17, Cl 16a de SEQ ID NO 18, Mo VIA de SEQ ID NO 19, MoVIB de SEQ ID NO 20, PnVIA de SEQ ID NO 21, PnVIB de SEQ ID NO 22, RsXXVIA de SEQ ID NO 23, T VIA de SEQ ID NO 24, CVIA de SEQ ID NO 25, CVIB de SEQ ID NO 26, CVIC de SEQ ID NO 27, CVID de SEQ ID NO 28, CVIE de SEQ ID NO 29, CVIF of SEQ ID NO 30, RVIA of SEQ ID NO 31 and F VIA of SEQ ID NO 32, or one of their variant(s), natural or synthetic, functionally active, exhibiting between 80% and 99% sequence homology.

5. Pain-relieving pharmaceutical composition according to any one of the preceding claims, characterized in that said active ingredient is a co-conotoxin MVIIA of sequence SEQ ID NO 1; or; one of its natural or synthetic, functionally active variants having between 80% and 99% sequence homology with SEQ ID NO 1.

6. Pain-relieving pharmaceutical composition according to any one of the preceding claims, characterized in that it comprises, in mass percentage, relative to its total mass, between 0.0002% and 6% of said co-conotoxin peptide(s), preferably between 0.0004% and 4.1%.

7. Pain-relieving pharmaceutical composition according to any one of the preceding claims, characterized in that it is in a pharmaceutically and dermatologically acceptable galenic form, for use by the cutaneous route and for topical action.

8. Pain-relieving pharmaceutical composition according to any one of the preceding claims, characterized in that it is in a galenic form of paste, ointment, cream, gel, balm, mousse, body milk, emulsion, solution, lotion or powder.

9. Pain-relieving pharmaceutical composition according to any one of the preceding claims, characterized in that it is administered by spreading, via a localized massage action, on the hands and / or feet and / or the painful area of ​​skin to be treated for pain, at least twice a day, preferably morning and evening, preferably with a therapeutically effective amount of between 0.1 mg and 1000 mg, preferably between 3.8 and 396 mg of co-conotoxin peptide(s) / liter of pharmaceutical composition.

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