Peptides and methods for treating pain

Abstract

The present invention relates to a set of peptides, compositions and kits comprising or allowing the expression of said peptides, and uses thereof, in particular for the prevention or treatment of pain in a subject in need thereof.

Description

Technical Field

[0001] The present invention relates generally to the field of pain management. More specifically, the present invention relates to a group of peptides, compositions and kits containing or allowing the expression of said peptides, and uses thereof, in particular for preventing or treating pain in a subject in need thereof. The group of peptides is a specific fragment of the human TAFA-4 protein or an equivalent fragment thereof, which can be used as an active ingredient for preventing or treating pain, in particular acute, subacute or chronic pain originating from neuropathic pain, postoperative pain or inflammatory pain, or for preventing and / or treating hyperalgesia or allodynia caused by pain caused by injury. Background Art

[0002] Pain is often classified as acute or chronic. Acute pain is short-lived and essential to maintaining the integrity of our bodies, whereas chronic pain persists beyond normal healing time and has adverse effects on health. Chronic inflammatory, neuropathic, or postoperative pain can cause long-lasting sensory abnormalities such as hyperalgesia (extreme pain caused by noxious stimuli) and mechanical allodynia (pain caused by innocuous mechanical stimuli). These pain categories differ in etiology and clinical features, but they share several common mechanisms, including alterations in neuroimmune interactions and neuronal sensitization (both peripheral and central) (Costigan et al., 2009). There is growing evidence that loss of inhibition may be a key mechanism underlying chronic pain (Bourane et al., 2015a; Bourane et al., 2015b; Boyle et al., 2019; Coullet et al., 2005; Duan et al., 2014; Peirs et al., 2015; Petitjean et al., 2019; Petitjean et al., 2015; Zeilhofer et al., 2012; Zhang et al., 2018, Yoo et al., 2021). However, despite our extensive understanding of the mechanisms and circuits underlying chronic pain in rodents, translation of these findings into effective treatments for chronic pain in humans remains elusive (Colloca et al., 2017). Indeed, nonsteroidal anti-inflammatory agents (NSAIDs) have limited efficacy for chronic pain, while opioids have multiple side effects, including potentially fatal respiratory depression, nausea, constipation, hyperalgesia, tolerance, and physical and psychological dependence (Benyamin et al., 2008). Therefore, efforts to identify new targets for the treatment of chronic pain with analgesic or analgesic potential should be encouraged.

[0003] In the past few years, the inventors have discovered significant features of the secreted protein TAFA-4 that suggest it could be an interesting drug for the treatment of chronic pain (WO2014180853). TAFA-4 belongs to a family of five highly conserved secreted neurokines (Sarver et al., 2021). TAFA-4 contains a signal peptide followed by a highly conserved core region with 10 cysteine ​​residues, including a CC-chemokine motif that makes it similar to a cytokine (Tom Tang et al., 2004).

[0004] The inventors have also previously demonstrated that in mice with TAFA-4 gene deleted, mechanical hypersensitivity induced by nerve injury lasts much longer than in wild-type (WT) mice (Delfini et al., 2013). These phenotypes can be reversed by intrathecal injection of recombinant TAFA-4.

[0005] As described in WO2014180853, the inventors previously reported experimental evidence that TAFA-4 has a powerful effect on pathologically increased mechanical pain. Studies have shown that intrathecal and subcutaneous administration of human recombinant TAFA-4 can reverse inflammatory, postoperative and nerve injury-induced mechanical hypersensitivity in male and female mice. They also showed that TAFA-4 can reverse neuronal sensitization of spinal lamina Ii interneurons caused by nerve injury, which are reported to be responsible for mechanical threshold changes. However, since TAFA-4 is a cysteine-rich protein (10 cysteines in total), the production of recombinant mature TAFA-4 protein can be challenging. In fact, refolding can lead to the acquisition of non-native conformations or inappropriate disulfide bridging patterns, which may greatly affect the activity of the protein. In addition, the several purification steps required to obtain high-purity recombinant TAFA-4 protein and possible aggregation problems can limit the yield of correctly folded protein. Furthermore, although chemical synthesis of TAFA-4 protein has been achieved, it is also challenging due to the length of the protein, requiring the assembly of no less than four (protected or partially protected) peptide segments (starting from the N-terminus and ending with the most C-terminal peptide). To generate full-length TAFA-4, three ligation and purification steps are required, resulting in low yields and being unsuitable for industrial-scale production. Therefore, alternative therapeutic agents and methods are urgently needed for the effective prevention or treatment of pain, especially acute, subacute or chronic pain, and it would be of great advantage to identify novel compounds with similar or better activity than the full-length TAFA-4 protein, which can be prepared on an industrial scale and in a cost-effective manner. SUMMARY OF THE INVENTION

[0007] The present invention is based at least in part on the identification of peptides that exhibit analgesic or analgesic activity, in particular analgesic activity. The inventors have found that these peptides are advantageously capable of reversing mechanical hypersensitivity induced by nerve injury, surgery or by injection of inflammatory agents (such as carrageenan), i.e., capable of treating neuropathic, postoperative or inflammatory pain.

[0008] The peptides of interest are or are derived from specific fragments of the human TAFA-4 protein. They are more effective than the full-length human TAFA-4 protein (also identified herein as "TAFA-4FL") for targeting pain, especially chronic pain, typically injury-induced pain, and are easier to produce. In fact, these peptides are short peptides that do not need to be linked to each other to produce a therapeutic effect. The inventors hereby provide patients with new tools that can alleviate the problem of addiction to pain treatment.

[0009] In particular, the inventors describe here for the first time a group of products or components, in particular a group of peptides, a group of nucleic acids encoding such peptides, a group of vectors and / or cells allowing the expression of such peptides, the peptides comprising or consisting of a first peptide and a second peptide, the first peptide being SEQ ID NO: 1 (CFPGQVAGTTRAQPSCVEASIVIQKWW) or a peptide having at least 90% identity with SEQ ID NO: 1 (also identified herein as a "variant", "peptide variant" or "peptide variant" of the sequence SEQ ID NO: 1), the second peptide being 2 (CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTK) or a peptide having at least 90% identity with SEQ ID NO: 2 (also identified herein as a "variant", "peptide variant" or "peptide variant" of the sequence SEQ ID NO: 2), wherein the (at least two) peptides are not connected to each other. The variants are generally functional variants, i.e. variants having the same (therapeutic) effect as described herein by the inventors for the peptides of SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] Also described herein are compositions comprising a peptide of sequence SEQ ID NO: 1 or a variant thereof (i.e., a peptide having at least 90% identity with SEQ ID NO: 1), and a peptide of sequence SEQ ID NO: 2 or a variant thereof (i.e., a peptide having at least 90% identity with SEQ ID NO: 2), wherein the (at least two) peptides are not connected to each other.

[0011] In particular aspects, similar to the peptides of SEQ ID NO: 1 and SEQ ID NO: 2, variants thereof described herein modulate the excitability of spinal cord interneurons, preferably spinal cord lamina IIi interneurons.

[0012] In a preferred aspect, the glutamine (Q) residue at position 13 ( / reference to the position shown in SEQ ID NO: 1) in the peptide of SEQ ID NO: 1 remains unchanged in the variant of the peptide of SEQ ID NO: 1. Similarly, the tyrosine (Y) residue at position 18 ( / reference to the position shown in SEQ ID NO: 2) in the peptide of SEQ ID NO: 2 also remains unchanged or is replaced by serine (S) in the variant of the peptide of SEQ ID NO: 2.

[0013] The inventors describe here in particular a group of nucleic acids allowing the expression of (at least two) peptides that are not linked to each other, as well as a composition comprising a group of such nucleic acids, wherein the (first) nucleic acid comprises a sequence encoding a peptide of SEQ ID NO:1 or a peptide that is at least 90% identical to SEQ ID NO:1, and the (second) nucleic acid comprises a sequence encoding a peptide of SEQ ID NO:2 or a peptide that is at least 90% identical to SEQ ID NO:2.

[0014] The present invention also describes a group of vectors allowing the expression of (at least two peptides) that are not linked to each other, as well as a composition comprising a group of such vectors, wherein the (first) vector comprises a sequence encoding a peptide of SEQ ID NO:1 or a peptide having at least 90% identity with SEQ ID NO:1, and the (second) vector comprises a sequence encoding a peptide of SEQ ID NO:2 or a peptide having at least 90% identity with SEQ ID NO:2.

[0015] Further disclosed herein are cells that allow expression of (at least two) peptides as described herein (or variants thereof) that are not linked to each other, and wherein i) the cell comprises a (first) nucleic acid sequence or vector encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity to SEQ ID NO: 1, and a (second) nucleic acid sequence or vector encoding a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity to SEQ ID NO: 2. Also disclosed are compositions comprising such cells and an acceptable support.

[0016] Also described herein are the groups, compositions or cells disclosed herein for the first time for use as active therapeutic or prophylactic ingredients, typically for use as pharmaceutical compositions, medicaments or medicines.

[0017] Any composition described herein is typically a pharmaceutical, therapeutic, veterinary or dietary / dietary composition comprising, in addition to the (at least two) peptides, a pharmaceutically and / or dietary / dietary acceptable support.

[0018] In a particular aspect, the composition may further comprise at least one additional (different) active compound / agent, preferably an agent effective against pain (acute, subacute or chronic pain), even more preferably a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID) or an opioid.

[0019] The present invention also relates to a group or cell as described herein, generally to a group of peptides, nucleic acids encoding such peptides or vectors allowing the expression of such peptides, or to a cell comprising or expressing such a group, for use [as an active ingredient] in the prevention or treatment of pain in a subject in need thereof. The present invention also relates to the use of such a peptide or such a group for the preparation of a pharmaceutical composition, a medicament or a drug for the prevention or treatment of pain in a subject in need thereof. In particular, the present invention can be used to treat chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia, allodynia or acute pain. The present invention can also be used to treat arthritis pain, pain associated with joint injuries, pain associated with Ehlers-Danlos syndrome, pain caused by sunburn or pain caused by ice burns.

[0020] The present invention also relates to a method for preventing or treating pain in a subject in need thereof, comprising administering a (therapeutically effective) amount of a product as described herein, such as a group and / or composition as described herein, preferably a group of peptides comprising: a peptide of SEQ ID NO: 1 or a variant thereof having at least 90% identity with SEQ ID NO: 1, and a peptide of SEQ ID NO: 2 or a variant thereof having at least 90% identity with SEQ ID NO: 2, or a composition comprising such a group.

[0021] The products, especially the peptides, groups and compositions described herein can be administered to the subject by any route, such as intramuscularly, intravenously, intraperitoneally, orally (orally), anally, transdermally, subcutaneously, topically, transdermally, transdermally or intrathecally. Preferably, the products (e.g., peptides or compositions) described herein are administered to the subject subcutaneously, orally or intravenously, even more preferably subcutaneously or orally.

[0022] Each product (eg, a peptide) or each composition described herein may be part of a kit.

[0023] A specific kit of the invention comprises: i) a first peptide or a nucleic acid sequence encoding such a peptide, the first peptide being SEQ ID NO: 1, a peptide having at least 90% identity to SEQ ID NO: 1, ii) a second peptide or a nucleic acid encoding such a peptide, the second peptide being SEQ ID NO: 2, a peptide having at least 90% identity to SEQ ID NO: 2, the first peptide and the second peptide or the nucleic acid encoding the peptide being in separate containers, and optionally iii) written instructions for using the kit. The kits described herein are generally used for preventing or treating pain.

[0024] In a specific aspect, the products of the invention can also be used in a research context. Nucleic acids encoding the peptides described herein or variants thereof, vectors allowing their expression, or cells comprising such nucleic acids or modified using such vectors can be used to express or modulate the expression of the following peptides in vitro or ex vivo: a peptide of SEQ ID NO: 1 or a functional variant thereof having at least 90% identity with SEQ ID NO: 1 and / or a peptide of SEQ ID NO: 2 or a functional variant thereof having at least 90% identity with SEQ ID NO: 2. In another specific aspect, the invention also relates to transgenic animals modified to express at least two different nucleic acids described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Comparison of the analgesic effect of TAFA-4 (full-length protein) and a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 when administered subcutaneously in vivo in the preserved nerve injury (SNI) model of neuropathic pain. Mice with mechanical hypersensitivity were subcutaneously administered a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (the group is identified here as "peptide 1 + 2"), TAFA-4 full-length protein (identified as "TAFA4 FL"), a negative control (vehicle) or a positive control (pregabalin). The figure shows the change in the mechanical force (equivalent to its weight in grams) required to induce a paw withdrawal reaction in 50% of the mice under each condition over time. The data are expressed as a percentage response relative to the baseline level. These measurements were performed before the implementation of the SNI pain model (T0 = baseline), 14 days after SNI to verify the establishment of mechanical allodynia, and then 1, 2 and 4 hours after subcutaneous injection. Statistical differences between “Peptide 1+2” treated mice, “TAFA-4FL” treated mice and vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001).

[0026] Figure 2: Comparison of the analgesic effect of TAFA-4 (full-length protein) and a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 when orally administered in vivo in the SNI model of neuropathic pain. Mice with mechanical hypersensitivity were orally administered a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (the group is identified here as "peptide 1 + 2"), TAFA-4 full-length protein (identified as "TAFA4 FL"), a negative control (vehicle) or a positive control (pregabalin). The figure shows the change in the mechanical force (equivalent to its weight in grams) required to cause 50% of the mice to withdraw their paws under each condition over time. The data are expressed as a percentage response relative to the baseline level. These measurements were performed before the implementation of the SNI pain model (T0 = baseline), 14 days after SNI to verify the establishment of mechanical allodynia, and then 1, 2 and 4 hours after oral gavage. Statistical differences between “Peptide 1+2” treated mice, “TAFA-4FL” treated mice and vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001).

[0027] Figure 3 : The analgesic effect of the two peptide groups of SEQ ID NO:1 and SEQ ID NO:2 when intravenously administered in vivo in the SNI model of neuropathic pain. Mice with mechanical hypersensitivity were intravenously administered with the two peptide groups of SEQ ID NO:1 and SEQ ID NO:2 (the group is identified as "peptide 1+2" here) or negative control (vehicle). The figure shows the change in the mechanical force (equivalent to its weight in grams) required to cause 50% of the mice to withdraw their paws under each condition over time. The data are expressed as a percentage response relative to the baseline level. These measurements were performed before the implementation of the SNI pain model (T0 = baseline), 14 days after SNI to verify the establishment of mechanical allodynia, and then 1, 2 and 4 hours after intravenous injection. Statistical differences between mice treated with "peptide 1+2" and the vehicle group are shown (two-way RM ANOVA, followed by Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001).

[0028] Figure 4 : Analgesic effect of a group of two peptides of SEQ ID NO: 1 and ID NO: NO2 when administered subcutaneously in vivo in a postoperative pain model (Brennan paw incision model) in male and female mice.

[0029] A. Schematic diagram of the protocol. Groups of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 or negative control (vehicle) were subcutaneously administered twice daily at different time points: one day before surgery (D-1), the day of surgery (D0), and D+1 and D+2 days after surgery. The administration of D0 was performed 1 hour before surgery and 1 hour after surgery (and waking up), respectively. Mechanical threshold response measurements were performed on D+1 day and D+2 before the administration of the two peptide groups or vehicle.

[0030] B. The graph represents the mechanical force (equivalent to its weight in grams) required to elicit a paw withdrawal reaction in 50% of the mice as a function of time. Mice treated with a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (this group is identified herein as "Peptide 1+2") were compared with mice treated with a negative control (vehicle). Statistical differences between mice treated with "Peptide 1+2" and the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: ***p<0.001). "Peptide 1+2"-treated group: 24 mice, vehicle group: 22 mice; Model = postoperative pain model; Gender: 12 males and 12 females for mice treated with "Peptide 1+2", 11 males and 11 females for the vehicle group; Administration: subcutaneously; Total concentration of "Peptide 1+2": 300 μg / kg. (approximately corresponds to Peptide 1: 128 μg / kg, for Peptide 2: 172 μg / kg)

[0031] Figure 5 : Repeated subcutaneous administration of the two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 did not induce tolerance in the chronic constriction injury (CCI) neuropathic pain model.

[0032] A. Schematic diagram of the protocol. After the CCI neuropathic pain model was established, mice were treated twice a day with a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (this group is referred to herein as "peptide 1 + 2") or a negative control (vehicle) for 14 consecutive days starting 7 days after surgery. The analgesic effect of "peptide 1 + 2" was determined every two days.

[0033] B. The graph represents the mechanical force (equivalent to its weight in grams) required to elicit a paw withdrawal reaction in 50% of the mice as a function of time. Mice treated with "Peptide 1 + 2" were compared with mice treated with a negative control (vehicle). Statistical differences between the analgesic effect after the first and last administration of "Peptide 1 + 2" treated mice and the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: ns: not significant). Model = CCI neuropathic pain model; Gender: 10 male mice for "Peptide 1 + 2" treatment, n = 9 for the vehicle group; Administration: subcutaneously; Total concentration of "Peptide 1 + 2": 300 μg / kg.

[0034] Figure 6 : Comparison of different therapeutic doses of a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 administered subcutaneously in vivo in a postoperative pain model (Brennan paw incision model) in male and female mice.

[0035] A. Schematic diagram of the protocol. According to three different protocols, groups of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("Peptide 1+2") or negative control (vehicle) were subcutaneously administered twice a day. "D-1 / D0" protocol: one day before surgery (D-1) and the day of surgery (D0); "D0" protocol: the day of surgery (D0); "D0 / D+1 / D+2" protocol: one day before surgery (D-1), the day of surgery (D0), and D+1 and D+2 days after surgery. The administration of D0 was performed 1 hour before surgery and 1 hour after surgery (and waking up), respectively. Mechanical threshold response measurements were performed on D+1 day and D+2 before the administration of the two peptide groups or the vehicle.

[0036] B. The graph represents the mechanical force (equivalent to its weight in grams) required to induce a paw withdrawal reaction in 50% of the mice as a function of time. Mice treated with "Peptide 1 + 2" were compared with mice treated with a negative control (vehicle) according to the three different protocols described above, "D-1 / D0", "D0", "D0 / D+1 / D+2". Statistical differences between mice treated with "Peptide 1 + 2" and the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: **p<0.01, ***p<0.001). "Peptide 1 + 2"-treated group: 16 mice; vehicle group: 11 mice; model = postoperative pain model; sex: 8 males and 8 females for mice treated with "Peptide 1 + 2", 6 males and 5 females for the vehicle group; administration: subcutaneously; total concentration of "Peptide 1 + 2": 300 μg / kg.

[0037] Figure 7: Analgesic effect of the group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 administered intravenously in vivo in an inflammatory pain model (capsaicin inflammatory pain model). The graph represents the mechanical force (equivalent to its weight in grams) required to cause a paw withdrawal reaction in 50% of mice in a function of time. Mice treated with a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (this group is identified herein as "peptide 1 + 2") were compared with mice treated with a negative control (vehicle). Statistical differences between mice treated with "peptide 1 + 2" and the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001). ; Model = inflammatory pain model; "peptide 1 + 2"-treated group: 8 mice Vehicle group: 8 male mice; Administration: subcutaneous; Total concentration of "peptide 1 + 2": 300 μg / kg.

[0038] Figure 8 : Analgesic effect of a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 administered intravenously in vivo in an osteoarthritis pain model (monoiodoacetate model).

[0039] The graph represents the mechanical force (equivalent to its weight in grams) required to elicit a paw withdrawal response in 50% of the mice as a function of time. Mice treated with a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (this group is identified herein as "Peptide 1+2") were compared to mice treated with a negative control (vehicle). Statistical differences between "Peptide 1+2" treated mice and the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001). ; Model = osteoarthritis pain model; "Peptide 1+2"-treated group: 8 mice, vehicle group: 8 male mice; Administration: subcutaneously; Total concentration of "Peptide 1+2": 300 μg / kg.

[0040] Fig. 9 : Analgesic effect of a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 administered intravenously in vivo in a frostbite pain model.

[0041] The graph represents the mechanical force (equivalent to its weight in grams) required to elicit a paw withdrawal reaction in 50% of the mice as a function of time. Mice treated with a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (this group is identified herein as "Peptide 1+2") were compared with mice treated with a negative control (vehicle). Statistical differences between "Peptide 1+2" treated mice and the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001). ; Model = frostbite pain model; "Peptide 1+2"-treated group: 8 mice, vehicle group: 8 male mice; Administration: subcutaneously; Total concentration of "Peptide 1+2": 300 μg / kg.

[0042] Fig.10 : Analgesic effect of a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 administered intravenously in vivo in an ultraviolet burn pain model.

[0043] The graph represents the mechanical force (equivalent to its weight in grams) required to elicit a paw withdrawal response in 50% of the mice as a function of time. Data are expressed as a percentage response to baseline levels. Mice treated with a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (the group is identified herein as "peptide 1 + 2") were compared with mice treated with a negative control (vehicle). Statistical differences between mice treated with "peptide 1 + 2" and the vehicle group are shown (two-way RM ANOVA followed by Bonferroni post hoc test: *p<0.05, **p<0.01, ***p<0.001). ; Model = UV burn pain model; 16 mice per group (8 male and 8 female mice); Administration: subcutaneous; Total concentration of "peptide 1 + 2": 300 μg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0045] definition

[0046] The terms used in this specification generally have their ordinary meaning in the art in the context of the present invention and in the specific context in which each term is used. Some terms are discussed below or elsewhere in the specification to provide additional guidance to the skilled reader in describing the methods of the present invention and how to use these methods. In addition, it is understood that the same item can be said in more than one way. Therefore, alternative language and synonyms can be used for one or more terms discussed herein. Synonyms for certain terms are provided. The description of one or more synonyms does not exclude the use of other synonyms.

[0047] The terms "peptide", "peptide variant", "protein fragment", "protein segment" are used interchangeably and refer to a sequence or polymer of amino acid residues. Such polymers of amino acid residues may contain natural or non-natural amino acid residues. The term also includes post-expression modifications of the peptide, such as glycosylation, sialylation, acetylation, amidation, phosphorylation, aminomethylation, etc. In addition, in the context of the present invention, "peptide" refers to a protein fragment or segment, which may include such post-expression modifications or a combination of these modifications, as well as optionally one or more modifications to the native (wild-type) sequence, such as deletions, additions and substitutions (usually conservative in nature), as long as the peptide retains the desired activity, i.e., preventing or treating pain. These modifications are preferably deliberate mutations, for example obtained by site-directed mutagenesis.

[0048] The term "isolated peptide" refers to a peptide that has been removed from its original environment (i.e., the natural environment if it occurs naturally). A peptide that occurs naturally in a natural system (e.g., in a living animal) is to be distinguished from the same peptide separated from all or part of the coexisting materials in the natural system. An isolated peptide is referred to herein as an "isolated peptide." In other words, in the context of the present invention, an isolated peptide is a fragment of a TAFA-4 protein that does not occur in nature.

[0049] The term "synthetic peptide" refers to a peptide obtained by chemical synthesis.

[0050] As used herein, the term "group of peptides" or "peptide group" refers to at least two peptides used in combination as (the group's) active ingredients, which may be mixed in the composition but not connected to each other. If there are two peptides, the peptides of the group will not be combined together by any bond (if the peptides of the group are more than two, the peptides will not be combined with each other), for example, by a bond / link composed of a peptide (amino acid sequence), or by a bond / link of chemical nature or both organic and chemical nature. The peptides of the group are isolated peptides, synthetic peptides or mixtures thereof. The group can be in the form of a dry powder, solution, suspension or colloid, and can be homogeneous or inhomogeneous. The group of peptides is usually part of a composition. The composition usually also includes a pharmaceutically and / or dietary acceptable support, carrier, excipient or vehicle. Typically, the excipient can be ascorbic acid and / or ascorbate. Ascorbic acid or L-ascorbic acid (IUPAC name: (5R)-5-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one) is a naturally occurring water-soluble vitamin commonly referred to as vitamin C. Examples of ascorbate salts include, but are not limited to, magnesium ascorbate, sodium ascorbate, calcium ascorbate, and ascorbyl palmitate. L-ascorbic acid and its sodium salt are antioxidants that can be used as preservatives or stabilizers when used in combination with the peptides of this group. Other antioxidants (e.g., methionine) may also be used.

[0051] The terms "sequence identity", "sequences having at least X% identity", and "X% identical sequences", where X is, for example, 90, are used interchangeably and refer to the degree to which sequences are identical on a nucleotide-to-nucleotide basis or an amino acid-to-amino acid basis over a comparison window. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence to achieve optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences or the number of positions where the nucleic acid base or amino acid residue is aligned with a gap to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. Preferably, the sequence identity is determined over the entire length of the reference sequence, here SEQ ID NO: 1 or SEQ ID NO: 2.

[0052] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA), or by visual inspection (see generally Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)).

[0053] Examples of algorithms suitable for determining sequence identity and sequence similarity percentages are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990), J. Mol. Biol. 215: 403-410 and Altschul et al., (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website. The algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length "W" in the query sequence, which match or meet a positive threshold score "T" when compared with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. Then, word hits are extended in both directions along each sequence until the cumulative comparison score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters "M" (reward score for a pair of matching residues; always > 0) and "N" (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction ceases when: the cumulative alignment score falls by the number "X" from its maximum achieved value; the cumulative score becomes zero or below due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation ("E") of 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0054] The degree of percent amino acid sequence identity can also be obtained by ClustalW analysis (version W1.8) by counting the number of identical matches in the alignment and then dividing the number of identical matches by the length of the reference sequence, and using the following default ClustalW parameters to achieve slow / exact pairwise optimal alignment - Gap opening penalty: 10; Gap extension penalty: 0.10; Protein weight matrix: Gonnet series; DNA weight matrix: IUB; Switch slow / fast pairwise alignment = slow or exact alignment.

[0055] As used herein, the terms "nucleic acid" or "polynucleotide" refer to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, these terms include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of a polynucleotide may comprise a sugar and phosphate group (commonly found in RNA or DNA), or a modified or substituted sugar or phosphate group. Alternatively, the backbone of a polynucleotide may comprise a polymer of synthetic subunits, such as phosphoamide, and may therefore be an oligodeoxynucleoside phosphoamide (P-NH 2 ) or mixed phosphoamido-phosphodiester oligomers. In addition, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing complementary strands and annealing the strands under appropriate conditions, or by de novo synthesis of complementary strands using a DNA polymerase and appropriate primers.

[0056] The terms "expression cassette", "nucleic acid construct" or "nucleic acid construct" are used interchangeably herein and refer to a nucleic acid comprising at least one transcription promoter operably linked to one or more (coding) sequences of interest (having a meaning understood by those skilled in the art), typically to an operon comprising several coding sequences of interest, the expression products of which contribute to the production of a function of interest in a cell, or to a nucleic acid further comprising an activation sequence and / or a transcription terminator.

[0057] The term "vector" refers to i) a DNA or RNA molecule containing a polynucleotide sequence (e.g., an expression cassette or a transgene) encoding a peptide, polypeptide, or protein, or ii) a product encapsulating such a DNA or RNA molecule. A vector generally contains a regulatory element capable of directing the expression of a coding polynucleotide sequence (also referred to as a transgene) in a cell into which a nucleic acid molecule is introduced. The term "transgene" refers to a polynucleotide that is introduced into a cell and can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In certain aspects, it imparts the desired characteristics to the cell into which it is introduced, or otherwise leads to a desired technical effect, which is generally a therapeutic effect. A transgene may contain a sequence encoding one or more proteins or one or more protein fragments. In a specific aspect, a vector is a plasmid. Alternatively, a vector is any well-known vehicle capable of artificially carrying an exogenous nucleic acid sequence of interest as described herein, such as, for example, a viral vector, a clay, a phage, or a double lipid membrane (e.g., a liposome) that encapsulates a product of interest as described herein and can be used to deliver it to a site of interest.

[0058] The term "gene therapy" refers to the treatment of a subject by delivering genes / nucleic acids into individual cells for the purpose of preventing or treating disease.

[0059] The term "transfection" refers to the uptake of foreign polynucleotides by a cell (e.g., a prokaryotic or eukaryotic cell). When an exogenous polynucleotide is introduced into a cell, the cell is identified as "transfected". Various transfection techniques are well known in the art. See, for example, Graham et al., Virology 52:456 (1973), Sambrook et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989), Davis et al., Basic Methods in Molecular Biology, Elsevier (1986), and Chu et al., Gene 13:197 (1981). Such techniques can be used to introduce one or more exogenous nucleic acids into a suitable host cell.

[0060] The term "transduction" refers to the delivery of a nucleic acid molecule into a recipient host cell, for example, by a gene delivery vector, such as a recombinant viral vector, particularly a retrovirus, adenovirus, recombinant adeno-associated virus (AAV), herpes simplex virus or lentivirus. For example, transduction of a target cell by rAAV virions results in the transfer of the rAAV vector contained in the virion into the transduced cell.

[0061] Recombinant "adeno-associated virus (AAV)" is a small Dependoparvovirus with a single-stranded linear DNA genome, lacking pathogenicity and low immunogenicity, which is artificially produced using a recombinant method. Recombinant AAV (rAAV) preferably has tissue / cell-specific targeting capabilities, so that the transgene of rAAV is specifically or preferentially delivered to one or more predetermined tissues / cells. The type and route of administration of AAV capsid and regulatory regions are important factors in determining these tissue-specific targeting capabilities.

[0062] In the context of the present invention, the term "pain" refers to any pain or sensitivity associated with tissue damage. Preferably, the term "pain" used herein is understood to be abnormal sensitivity, i.e., a hypersensitivity reaction mediated by abnormally enhanced activity of all types of sensory neurons (including nociceptors and non-nociceptors). The term pain includes any pain selected from the group consisting of nociceptor-mediated pain (also referred to herein as "nociceptive pain"), neuropathic pain, inflammatory pain, pathological pain, acute pain, subacute pain, chronic pain, mechanical pain, chemical pain, somatic pain, visceral pain, deep somatic pain, superficial somatic pain, somatoform pain, allodynia, hyperalgesia, or pain associated with nerve damage.

[0063] "Nociceptive" pain or "nociceptor-mediated" pain occurs as a result of intense or noxious stimulation activating a specific subset of peripheral sensory neurons (nociceptors). According to the present invention, nociceptive pain includes mechanical pain (squeezing, tearing, etc.) and chemical pain (iodine in a wound, chili powder in the eye). Examples of nociceptive pain include, but are not limited to, traumatic or surgical pain, labor pain, sprains, fractures, burns, bruises, abrasions, injections, dental surgery, skin biopsies, and obstructions. Nociceptive pain includes visceral pain and somatic pain, particularly deep somatic pain and superficial somatic pain.

[0064] "Visceral pain" is diffuse, difficult to localize, and typically involves distant, often superficial structures. It may be accompanied by nausea and vomiting, and may be described as nauseating, deep, squeezing, and / or dull. Deep somatic pain is caused by the stimulation of nociceptors in ligaments, tendons, bones, blood vessels, fascia, and muscles, and is a dull and poorly localized pain. Examples of deep somatic pain include sprains and fractures. Superficial pain is caused by the activation of nociceptors in the skin or other superficial tissues, and is sharp, well-defined, and well-localized. Examples of injuries that produce superficial somatic pain include minor wounds and minor (first-degree) burns.

[0065] In the context of the present invention, the term "injury-induced pain" encompasses neuropathic pain, inflammatory pain and postoperative pain.

[0066] Inflammatory pain is pain that occurs in the presence of tissue damage or inflammation, including postoperative pain, post-traumatic pain, arthritis (rheumatoid arthritis or osteoarthritis) pain, pain associated with autoimmune diseases (such as psoriasis), and pain associated with joint, muscle and tendon injuries, such as axial low back pain. Inflammation causes sensitization of peripheral sensory neurons, causing spontaneous pain and rendering pain hypersensitivity ineffective. Acute or chronic pathological tissue inflammation strongly affects pain perception through sensitization of peripheral sensory neurons, causing local and disabling pain hypersensitivity. It is known that inflammatory mediators can enhance the excitability of noxious primary afferent fibers in part by changing the expression and / or function of ion channels present in nerve endings.

[0067] "Neuropathic pain" is a common chronic non-malignant pain that is caused by injury or dysfunction of the peripheral or central nervous system. Neuropathic pain can have different etiologies and can occur due to, for example, trauma, surgery, herniated disc, spinal cord injury, diabetes, herpes zoster infection (shingles), HIV / AIDs, advanced cancer, amputation (including mastectomy), carpal tunnel syndrome, long-term alcohol consumption, exposure to radiation, and unexpected side effects of neurotoxic therapeutic drugs (such as certain anti-HIV and chemotherapy drugs). A specific type of neuropathic pain is "chemotherapy-induced peripheral neuropathic pain" (CIPN) or "chemotherapy-induced neuropathic pain" (CINP). CINP or CIPN is one of the most serious side effects of anticancer drugs such as platinum and taxane-derived drugs (oxaliplatin, cisplatin, carboplatin and paclitaxel). CINP can even be a factor in discontinuing treatment, thereby increasing the risk of death. Neuropathic pain is often characterized by or results from the development of chronic allodynia (defined as pain caused by stimuli that do not normally evoke a painful response, such as light touch) and / or hyperalgesia (defined as increased sensitivity to normally painful stimuli) and may persist for months or years after any damaged tissue has apparently healed. Patients with cancer may also experience pain, which may result from a variety of causes, including inflammation, compression, invasion, metastatic spread to bone or other tissues. Pain also includes migraines and headaches related to activation of sensory fibers innervating the meninges.

[0068] The products of the present invention, in particular the peptides, groups, cells or compositions, can be used to prevent or treat chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia, allodynia or acute pain.

[0069] In another specific embodiment, the product of the present invention, in particular the peptide, group, cell or composition, can be used to prevent or treat arthritis pain, pain associated with joint damage, pain associated with Ehlers-Danlos syndrome, pain caused by sunburn or pain caused by ice burns.

[0070] Specifically, the products of the invention, in particular the peptides, groups, cells or compositions, can be used for the prevention or treatment, in particular the treatment of pain associated with Ehlers-Danlos syndrome, which includes chronic muscle and / or bone and / or joint pain. The products of the invention, in particular the peptides, groups, cells or compositions, can also be used for the prevention or treatment, in particular the treatment of pain caused by sunburn (i.e. caused by overexposure to ultraviolet radiation) or pain caused by ice burns (also known as freezer burns or frostbite).

[0071] Preferably, the product of the present invention, in particular the peptide, group, cell or composition, is used to prevent or treat pain caused by injury. More preferably, the product of the present invention, in particular the peptide, group, cell or composition, is used to prevent or treat neuropathic pain (e.g., chemotherapy-induced neuropathic pain or chemotherapy-induced peripheral neuropathic pain), postoperative pain and / or inflammatory pain.

[0072] Typically, the product of the present invention, in particular the peptide, group, cell or composition, is used to prevent or treat pain caused by chronic injury. Typically, the product of the present invention is used to prevent or treat chronic neuropathic pain, such as chronic chemotherapy-induced peripheral neuropathic pain (CIPN), chronic chemotherapy-induced neuropathic pain (CINP) or pain caused by chronic nerve injury; chronic postoperative pain; and / or chronic inflammatory pain; chronic pain associated with or caused by severe burns.

[0073] In the context of the present invention, the term "treatment" or "treating" pain in a subject refers to delaying, stabilizing, curing, healing, alleviating, relieving, altering, improving, ameliorating, remedying or affecting any form of pain in a subject as described herein, or any disease or condition associated with pain, in particular acute, subacute or chronic pain (in particular any neuropathic condition associated with chronic pain typically caused by neuropathic pain, postoperative pain or inflammatory pain), or any symptom of such a disease or condition, after the subject is exposed to a suitable product or group of products of the present invention, i.e., applying or administering a suitable product or group of products of the present invention, in particular i) a group of peptides (wherein the peptides are not linked to each other), preferably sequences SEQ ID NO: 1 and SEQ ID NO: NO:2 or its variants, as defined herein, ii) a nucleic acid or a group of nucleic acids allowing the expression of such a group of non-linked peptides, iii) an expression cassette or a group of expression cassettes comprising a group of nucleic acids as defined herein, iv) a vector or a group of vectors allowing the expression of a group of non-linked peptides, v) a cell or a group of cells allowing the expression of the group of non-linked peptides, or vi) a composition according to the invention comprising such peptides, nucleic acids, expression cassettes, vectors and / or cells.

[0074] The term "treatment" or "treating" also refers to any indicator of success in treating pain (which may be associated with any injury, pathology or condition), including any objective or subjective parameter, such as reduction, alleviation, slowing of progression or severity, stabilization, reduction of pain symptoms, or making the subject tolerable or more tolerable pain. The term "treating" pain also includes increasing pain tolerance and / or reducing perceived pain. In particular aspects, the methods, compounds and compositions of the present invention are used to increase pain tolerance and / or reduce perceived pain. The term "pain tolerance" as used herein refers to the amount of pain that a subject can perceive and withstand before emotional and / or physical collapse. Pain tolerance is different from the pain threshold (the minimum mechanical stimulus required to produce pain). "Increasing pain tolerance" as used herein generally refers to a situation in which a subject can develop greater pain tolerance (i.e., less perceived pain) compared to a previous state, for example, after administering to a subject a suitable peptide of the sequence SEQ ID NO: 1 and SEQ ID NO: 2 or a variant thereof or a composition comprising the peptide or variant.

[0075] In the context of the present invention, "preventing" or "prevention" in relation to pain in a subject means at least reducing the likelihood that the subject will acquire (or be susceptible to acquire) any kind of pain, after exposure of the subject to a product of the present invention, i.e. application or administration of a suitable product or group of products of the present invention, in particular i) a group of suitable peptides (wherein the peptides are not linked to each other), preferably peptides of sequence SEQ ID NO: 1 and SEQ ID NO: 2 or variants (usually functional variants) thereof, as defined herein, ii) a nucleic acid or group of nucleic acids allowing the expression of such a group of peptides not linked to each other, iii) an expression cassette or group of expression cassettes comprising a group of nucleic acids as defined herein, iv) a vector or group of vectors allowing the expression of a group of peptides not linked to each other, v) a cell or group of cells allowing the expression of a group of peptides not linked to each other, or vi) a composition according to the present invention expressing such a peptide, nucleic acid, expression cassette, vector and / or cell. For example, "prevention" includes the absence of at least one clinical symptom of pain in a subject who may be exposed to or susceptible to pain but has not yet experienced or displayed symptoms of pain.

[0076] In the context of the present invention, "subject" or "patient" refers to an animal, particularly a mammal, in need of treatment for a disease or condition or its symptoms. The subject may be a subject who has been diagnosed with, or determined to be at risk of, a disease or condition that is known to cause pain in the subject. In a specific example, the subject is diagnosed with or suffers from pain, such as acute pain and / or subacute pain or chronic pain, including neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia and / or allodynia.

[0077] In certain aspects, the subject is a human.

[0078] In another specific aspect, the subject is an animal, particularly a domestic animal or breeding animal, particularly a horse, dog, cat, cow, or the like.

[0079] In another specific aspect, the subject has at least one mutant allele in the myo1A gene.

[0080] Peptides

[0081] The inventors have identified herein a group of novel components, in particular a group of peptides (wherein the peptides are not linked to each other), which when used in combination simultaneously or sequentially can prevent or treat pain, preferably simultaneously, and in particular can reverse mechanical hypersensitivity in the case of injured or inflamed nervous systems. Without being bound by any theory, the inventors believe that these peptides exhibit analgesic or analgesic activity by modulating the excitability of the spinal cord network, in particular by specifically targeting mechanically and / or chemically induced nociceptive signals.

[0082] The peptides described herein consist of a specific fragment of 105 amino acid residues from the mature protein of human TAFA-4 (SEQ ID NO 5: SQHLRGHAGHHQIKQGTCEVVAVHRCCNKNRIEERSQTVKCSCFPGQVA GTTRAQPSCVEASIVIQKWWCHMNPCLEGEDCKVLPDYSGWSCSSGNK VKTTKVTR) (generated by cleavage of the signal sequence and identified in public databases such as Tang et al., 2004, accession number #NP_0011005527 or Genbank accession number AAP92409).

[0083] The inventors describe here for the first time in particular a group of products / components, in particular a group of isolated or synthetic peptides, comprising, consisting of or consisting essentially of: i) a first peptide, which is SEQ ID NO: 1 (CFPGQVAGTTRAQPSCVEASIVIQKWW, i.e., Cys-Phe-Pro-Gly-Gln-Val-Ala-Gly-Thr-Thr-Arg-Ala-Gln-Pro-Ser-Cys-Val-Glu-Ala-Ser-Ile-Val-Ile-Gln-Lys-Trp-Trp) or a peptide having at least 90% identity to SEQ ID NO: 1 (referred to herein as a "variant", "peptide variant" or "peptide variant" of sequence SEQ ID NO: 1), and ii) a second peptide, which is SEQ ID NO: 1 NO:2 (CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR, i.e., Cys-His-Met-Asn-Pro-Cys-Leu-Glu-Gly-Glu-Asp-Cys-Lys-Val-Leu-Pro-Asp-Tyr-Ser-Gly-Trp-Ser-Cys-Ser-Ser-Gly-Asn-Lys-Val-Lys-Thr-Thr-Lys-Val-Thr-Arg) or a peptide having at least 90% identity with SEQ ID NO:2 (referred to herein as a “variant”, “peptide variant” or “peptide variant” of sequence SEQ ID NO:2), wherein the (at least two) peptides are not connected to each other.

[0084] Peptides having at least 90% identity to a particular amino acid / peptide sequence are referred to herein as "variants," "peptide variants," or "peptide variants" of said sequence. In preferred aspects, any variant present in the group or in a composition comprising / expressing such a group is a functional variant, as further explained below.

[0085] The terms "consisting essentially of" have the meanings usually ascribed to them, e.g., they allow for elements not explicitly recited, but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the invention. Thus, a group of peptides "consisting essentially of a particular sequence" refers to peptides that contain or consist of that sequence (the sequences are not contiguous to each other), and which include other features that are not essential for the activity of the peptide.

[0086] The peptide of SEQ ID NO: 1 consists of the 27 C-terminal amino acid residues of the human TAFA-4 mature protein and can be identified as amino acids 78-104 of UniProt accession number #Q96LR4.

[0087] The peptide of SEQ ID NO: 2 consists of the 36 C-terminal amino acid residues of the human TAFA-4 mature protein and can be identified as amino acids 105-140 of UniProt Accession No. #Q96LR4.

[0088] In particular aspects, the peptide of SEQ ID NO: 1 and / or the peptide of SEQ ID NO: 2 is acetylated at the N-terminal position and / or amidated at the C-terminal position.

[0089] The two amino acid residues Q (Glu) shown in bold at position 13 in SEQ ID NO:1:CFPGQVAGTTRAQPSCVEASIVIQKWW, and Y (Tyr) shown in bold at position 18 in SEQ ID NO:2:CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR (refer to the positions listed in SEQ ID NO 1 and SEQ ID NO 2, respectively), which the inventors believe are related to the biological activity of the peptide.

[0090] Variants of the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 are also disclosed by the inventors for the first time herein. A variant refers to a peptide derived from a peptide of SEQ ID NO: 1 or a peptide derived from a peptide of SEQ ID NO: 2, and is produced by deleting or adding one or more amino acids and / or replacing one or more amino acids at one or more sites in the peptide of SEQ ID NO: 1 or SEQ ID NO: 2. If the variant substantially retains the functional activity of the peptide of SEQ ID NO: 1 or SEQ ID NO: 2, or even improves the functional activity to allow the prevention or treatment of pain, the variant will be considered a functional variant. Activity can be measured using a functional assay (e.g., a behavioral assay performed in the experimental section).

[0091] Peptide variants useful in the context of the present invention may have at least 90%, such as (at least) 90.5%, 91%, 92%, 92.1%, 93%, 93.7%, 94%, 95%, 95.2%, 96%, 96.8%, 97%, 98%, 98.4% or 99% sequence identity as described above with the sequence encoding the peptide (e.g. SEQ ID NO: 1 or SEQ ID NO: 2).

[0092] In a particular aspect, a variant having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2 modulates the excitability of spinal interneurons (preferably spinal lamina IIi interneurons). In a preferred aspect, the variant is a peptide comprising one or more point mutations (e.g., 2, 3, 4, 5 or 6 point mutations) that add, delete or replace any amino acid present in the peptide sequence of interest as described herein (e.g., in particular, SEQ ID NO: 1 or SEQ ID NO: 2).

[0093] Specifically for peptide variants having at least 90% identity with SEQ ID NO:1 or SEQ ID NO:2, the previous paragraph applies, provided that the amino acid residue Q at position 13 in SEQ ID NO:1 remains unchanged in the amino acid sequence of the peptide having at least 90% identity with SEQ ID NO:1, and provided that the amino acid residue Y at position 18 in SEQ ID NO:2 remains unchanged or is substituted with serine (S) in the amino acid sequence of the peptide having at least 90% identity with SEQ ID NO:2 (positions 13 and 18 refer to the positions shown in SEQ ID NO:1 and SEQ ID NO:2, respectively). The peptide with amino acid residue S at position 18 is SEQ ID NO: 6 (CHMNPCLEGEDCKVLPDSSGWSCSSGNKVKTTKVTR, i.e., Cys-His-Met-Asn-Pro-Cys-Leu-Glu-Gly-Glu-Asp-Cys-Lys-Val-Leu-Pro-Asp-Ser-Ser-Gly-Trp-Ser-Cys-Ser-Ser-Gly-Asn-Lys-Val-Lys-Thr-Thr-Lys-Val-Thr-Arg).

[0094] Therefore, in a preferred aspect of the present invention, the amino acid Q (glutamine residue) at position 13 in the peptide of SEQ ID NO: 1 ( / with reference to the position shown in SEQ ID NO: 1) remains unchanged in the amino acid sequence of a variant of the peptide having at least 90% identity with SEQ ID NO: 1. Similarly, in another preferred aspect, the amino acid Y (tyrosine residue) at position 18 in the peptide of SEQ ID NO: 2 ( / with reference to the position shown in SEQ ID NO: 2) also remains unchanged or is substituted with (serine) in the amino acid sequence of a variant of the peptide having at least 90% identity with SEQ ID NO: 2.

[0095] In another preferred aspect of the present invention, the amino acid Q at position 13 (refer to SEQ ID NO: 1) remains unchanged in the amino acid sequence of a variant of a peptide having at least 90% identity with SEQ ID NO: 1, and the amino acid Y at position 18 (refer to SEQ ID NO: 2) also remains unchanged or is substituted with serine (S) in the amino acid sequence of a variant of a peptide having at least 90% identity with SEQ ID NO: 2.

[0096] In another specific aspect, a variant of the peptide of SEQ ID NO: 1 that can be used in the context of the present invention may have at least 88.8%, 92.5%, 96.2% sequence identity with the peptide sequence of SEQ ID NO: 1, preferably with the conditions described above with reference to the amino acid residue Q at position 13 of SEQ ID NO: 1. In another specific aspect, a variant of the peptide of SEQ ID NO: 2 that can be used in the context of the present invention may have at least 91.6%, 94.4%, 97.2% sequence identity with the peptide sequence of SEQ ID NO: 2, preferably with the conditions described above with reference to the amino acid residue Y at position 18 of SEQ ID NO: 2. Preferably, the variant modulates the excitability of spinal interneurons, preferably spinal lamina IIi interneurons.

[0097] In a specific aspect, a variant is a peptide comprising one or more point mutations (e.g., 1, 2 or 3 point mutations) that add, delete or replace any amino acid present in a peptide sequence of interest as described herein (e.g., in particular, SEQ ID NO: 1 or SEQ ID NO: 2), preferably with the conditions described above with reference to amino acid residue Q at position 13 of SEQ ID NO: 1 and amino acid residue Y at position 18 of SEQ ID NO: 2.

[0098] In a particular aspect, one or more deletions are located at the N-terminus of the sequence of interest (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). In another aspect, one or more deletions are at the C-terminus, or at any other position of the sequence of interest (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), with the restriction described above for SEQ ID NO: 1 and SEQ ID NO: 2 being related to the amino acid residue Q at position 13 with reference to SEQ ID NO: 1, and the amino acid residue Y at position 18 with reference to SEQ ID NO: 2. In yet another aspect, two or more deletions are located at the N-terminus and C-terminus of the sequence of interest (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). For example, such deletions at the N- and / or C-terminus or the core of a peptide of SEQ ID NO: 1 or SEQ ID NO: 2 can produce a truncated human peptide variant of SEQ ID NO: 1 or SEQ ID NO: 2.

[0099] In addition, any peptide in the group of peptides according to the present invention can be fused with another peptide or protein to form a conjugate that can be used in the methods described herein, as long as the "another peptide or protein" is not one of the "at least two" peptides in the group of the present invention. In fact, the peptides of the group of peptides described herein (the active ingredients of the group) should not be connected to each other. The term "conjugate" herein refers to an engineered fusion construct that combines the biological functions of two molecules in a single polypeptide, in particular a peptide of SEQ ID NO: 1 or 2 or a variant thereof, such as a polypeptide that is capable of regulating the excitability of nociceptors or interneurons, and, for example, a polypeptide that specifically interacts or binds to a target cell.

[0100] The present invention also relates to nucleic acids encoding peptides in the group of the present invention, such as peptides of sequence SEQ ID NO: 1, peptides of sequence SEQ ID NO: 2, or variants thereof as described herein. For example, the present invention encompasses any sequence encoding a peptide of SEQ ID NO: 1 or a peptide of SEQ ID NO: 2, or variants thereof, and similar sequences resulting from the degeneracy of the genetic code. In specific aspects, the nucleic acids encoding the peptides described herein comprise, consist of, or consist essentially of the sequence of SEQ ID NO 3: TGCTTCCCGGGACAGGTGGCGGGCACAACTCGGGCTCAACCTTCTTGT GTTGAAGCTTCCATTGTGATTCAGAAATGGTGG or SEQ ID NO 4: TGTCACATGAATCCGTGTTTGGAAGGAGAGGATTGTAAAGTGCTGCCA GATTACTCAGGTTGGTCCTGTAGCAGTGGCAATAAAGTCAAAACTACGAAGGTAACGCGG.

[0101] The inventors describe herein in particular a set of nucleic acid molecules comprising (at least two) peptides that allow expression that are not linked to each other, wherein the (first) nucleic acid comprises a peptide encoding a (first) peptide of interest (e.g., a peptide of SEQ ID NO: 1) or a peptide having at least 90% identity to SEQ ID NO: 1, and the (second) nucleic acid comprises a peptide encoding a (second) peptide of interest (e.g., a peptide of SEQ ID NO: 2) or a peptide having at least 90% identity to SEQ ID NO: 2. The inventors also describe herein a composition comprising a set of such nucleic acids, preferably together with an acceptable support, carrier, excipient or vehicle as described herein.

[0102] The above-mentioned nucleic acid sequence encoding the peptide of SEQ ID NO: 1, SEQ ID NO: 2 or a variant thereof may be adjacent to a regulatory sequence to control its expression in an appropriate host cell.

[0103] In another aspect, the present invention also relates to an expression cassette comprising, from 5' to 3', in the following order:

[0104] - promoter,

[0105] - a nucleic acid sequence encoding a peptide of interest (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof as described herein); and

[0106] - a termination signal, such as a polyadenylation signal.

[0107] The above-mentioned nucleic acid sequence encoding the peptide of interest or the expression cassette disclosed herein may be flanked by sequences suitable for packaging it into a vector that optimizes its transcription and / or translation in a cell.

[0108] In another aspect, the present invention also relates to vectors allowing the expression of a peptide or a set of peptides as described herein. The present invention also relates to vectors comprising a set of nucleic acids allowing the expression of (at least two) peptides not linked to each other, or a set of expression cassettes as defined herein.

[0109] In a further aspect, the inventors relate to a group of vectors that allow the expression of (at least two peptides) that are not linked to each other, wherein the (first) vector comprises a sequence encoding a (first) peptide of interest (e.g., a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1), and the (second) vector comprises a sequence encoding another (second) peptide of interest (e.g., a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2). The inventors also describe herein a composition comprising a group of such vectors, preferably together with an acceptable support, carrier, excipient or vehicle as described herein.

[0110] Method for producing the peptide

[0111] In order to allow expression in a host cell, the nucleic acid encoding one or more peptides described herein (or variants thereof) may be present in one or more vectors, and after the vector is introduced into a suitable host cell, the sequence may be expressed according to standard cloning and expression techniques known in the art to produce the encoded peptides described herein (e.g., as described in Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Various expression vectors may be used to express the polynucleotide sequences encoding the peptides described herein. The expression vectors that can be used in the present invention do not completely include eukaryotic expression vectors, in particular mammalian expression vectors, virus-based expression vectors, baculovirus expression vectors, plant expression vectors, and any plasmid expression vectors, so as to produce any of the peptides described herein in a host cell. The expression vector may also be a vector that allows the expression of peptides in a bacterial system. When a single nucleic acid encodes several peptides or a single vector allows the expression of several peptides, the resulting peptides are not connected to each other.

[0112] The choice of expression vector depends on the intended host cell in which the vector will be expressed. One skilled in the art can easily make this choice. The present invention also relates to cells, in particular host cells, comprising nucleic acid sequences encoding the peptides described herein. The inventors have also disclosed for the first time (host) cells modified using the vectors described herein.

[0113] Disclosed herein in particular are cells that allow expression of (at least two) peptides as described herein (or variants thereof) that are not linked to each other. The cell typically comprises a (first) nucleic acid sequence or vector encoding a peptide of interest (e.g., a peptide of SEQ ID NO: 1 or a peptide with at least 90% identity to SEQ ID NO: 1), and a (second) nucleic acid sequence or vector encoding a (second / different) peptide of interest (e.g., a peptide of SEQ ID NO: 2 or a peptide with at least 90% identity to SEQ ID NO: 2). Also disclosed are compositions comprising such cells, preferably together with an acceptable support, carrier, excipient or vehicle as described herein.

[0114] The present invention particularly discloses a group of cells that allow expression of (at least two) peptides (or variants thereof) as described herein that are not connected to each other. Each cell generally comprises a nucleic acid sequence or a vector encoding a peptide of interest, and the peptide of interest is, for example, a peptide of SEQ ID NO: 1 or SEQ ID NO: 2, or a peptide with at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2. Compositions comprising such cells are also disclosed, preferably together with an acceptable support, carrier, excipient or vehicle as described herein.

[0115] The peptides of the present invention or variants thereof can be expressed in prokaryotic or eukaryotic host cells. Representative host cells include many E. coli strains, mammalian cell lines, such as CHO, CHO-K1, and HEK293; insect cells, such as Sf9 cells; and yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris.

[0116] The nucleic acids or vectors described herein can be transfected into host cells by standard techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Alternatively, the nucleic acids or vectors described herein can be delivered into host cells by transduction using virus-based vectors.

[0117] Purity can be measured by any appropriate standard method, such as by column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis. An "isolated" or "synthesized" peptide also defines a degree of sterility for safe administration to human subjects, e.g., absence of infectious or toxic agents.

[0118] The peptides used in the compositions and methods described herein can also be produced by solid phase synthesis techniques. Direct chemical synthesis of peptides can be accomplished by methods well known to those skilled in the art, such as native chemical ligation (NCL). This chemical method consists of coupling unprotected peptide fragments: a peptide with an N-terminal cysteine ​​reacts with a C-terminal thioester peptide. This transthioesterification reaction is followed by rapid intramolecular S, N-acyl transfer, resulting in the formation of a native amide bond at the site of attachment.

[0119] Gene therapy

[0120] By cloning the nucleic acid sequence encoding the peptides described herein into an appropriate vector, the inventors also provide new gene delivery tools, in particular genetic constructs (e.g., expression cassettes or vectors) for gene therapy of pain, in particular acute, subacute or chronic pain, preferably chronic pain. Gene therapy can be used to allow specific cells in a subject to endogenously produce TAFA-4-peptides or variants thereof. Gene therapy can be performed in vivo or in vitro. In vitro gene therapy requires the separation and purification of at least a cell sample of the subject, the introduction of at least one nucleic acid sequence encoding the group of peptides described herein (i.e., transgene) into the isolated cells, and the genetically altered / modified cells are reintroduced into the subject. In contrast, in in vivo gene therapy, the transgene is usually packaged and administered to the subject. The gene delivery construct can be non-viral or viral. Preferably, the genetic construct described herein is prepared with viral elements, viral vectors and / or any viral packaging system, which can be used to express the transgene / coding sequence (i.e., the nucleic acid sequence encoding the group of peptides of interest or variants thereof) in the target tissue / cell. The viral vector can incorporate any suitable promoter and other transcriptional regulators to allow or promote the expression of the transgenic product in the target tissue / cell. The viral packaging system is preferably adapted to the target cell. Once inside the target cell, the system facilitates delivery to the target tissue. The viral vectors useful in the methods described herein are preferably replication-defective viruses, such as, for example, adenovirus or adeno-associated virus (AAV) vectors.

[0121] In some aspects, the present invention relates to a recombinant adeno-associated virus (AAV) comprising in the genome a nucleic acid sequence encoding at least one peptide, preferably at least two peptides, such as a peptide of SEQ ID NO: 1 and / or a peptide of SEQ ID NO: 2, or a variant thereof, of the group of interest. The coding portion of the nucleic acid is usually operably linked to a promoter.

[0122] To date, at least dozens of different AAV serotypes with different surface properties have been isolated and characterized from humans or non-human primates (NHPs). The term "serotype" allows the skilled artisan to distinguish between AAVs with serologically different capsids. Serological specificity is determined by the lack of cross-reactivity of antibodies to one AAV serotype compared to other AAV serotypes. The rAAV described herein, also referred to as rAAV vectors or rAAV particles, can have any of the following known serotypes, i.e., can be selected from, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV. Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68. rAAV vectors may have enhanced tropism for specific cells, tissues, or organs. In the case of administration by the oral route, the rAAV vector may have an enhanced tropism for gastric, small intestinal or colonic tissues, more specifically, for cells constituting these tissues, particularly epithelial cells such as enterocytes, goblet cells, enteroendocrine cells, Paneth cells or tuft cells. For target cells located in the intestine or delivered to the intestine, any of AAV4, AAV7, AAV8, AAV9 or AAV10 may be selected as being particularly effective.

[0123] The gene therapy vectors or cassettes of the present invention can be produced by methods well known to those skilled in the art and previously described in, for example, PCT Patent Application Publication No. WO03042397 and US Pat. No. 6,632,670.

[0124] The present disclosure also relates to a method for producing a recombinant vector (e.g., a viral vector such as AAV), comprising:

[0125] a) culturing cells that have been transfected / transduced with the recombinant vector described herein; and

[0126] b) Recovering the recombinant vector from the supernatant of the transfected / transduced cells.

[0127] The gene therapy vector of the present invention can be produced by transfecting two or three plasmids into 293 or 293T human embryonic kidney cell lines. In a specific aspect, the DNA encoding the therapeutic peptide is provided by one plasmid; the capsid protein and replication gene produced by one or more serotypes of AAV, and the auxiliary functions produced by adenovirus, for example, are all provided in trans by a second plasmid. In another specific aspect, the DNA encoding the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 or variants thereof is provided by one plasmid; the capsid protein and replication gene produced by one or more serotypes of AAV are provided in trans by a second plasmid, and the auxiliary functions produced by adenovirus, for example, are provided by a third plasmid. In a specific aspect, the first plasmid comprises an expression cassette comprising a nucleic acid sequence encoding a peptide of SEQ ID NO: 1 and a peptide of SEQ ID NO: 2 or variants thereof as described herein, the nucleic acid sequence being operably linked to a promoter, including two flanking inverted terminal repeats (ITRs). In another specific aspect, DNA encoding each therapeutic peptide is provided via two different plasmids, the first plasmid comprising a nucleic acid sequence encoding the peptide of SEQ ID NO: 1 or a variant thereof, and the second plasmid comprising a nucleic acid sequence encoding the peptide of SEQ ID NO: 2 or a variant thereof.

[0128] After cell culture, the gene therapy vector can be released from the cells, for example, by freeze-thaw cycles, and purified by any method known to those skilled in the art, such as by using an iodixanol step gradient followed by ion exchange chromatography on a Hi-Trap QHP column. The resulting gene therapy vector can then be concentrated by spin column, and the purified vector can be stored frozen (at -60°C or below), for example in phosphate buffered saline.

[0129] Related aspects of the invention include host cells transfected or transduced with at least one recombinant vector, such as host cells transduced with at least one AAV vector as described herein; or a group of cells transfected with a vector or vector group comprising DNA encoding a therapeutic peptide or variant thereof as defined herein. Further related aspects of the invention include any nucleic acid molecule comprising the genome of a recombinant vector (e.g., AAV) as described herein or (essentially) consisting thereof.

[0130] Composition

[0131] The compositions described herein comprise a group of (at least two) peptides as described herein that are not linked to each other, or a product as described herein that allows the expression of the (at least two) peptides (e.g., nucleic acids, cells, groups of nucleic acids or groups of cells), and may be pharmaceutical, therapeutic, prophylactic or veterinary compositions. The compositions may also be (possibly at the same time) dietary / dietary compositions.

[0132] The specific composition described herein comprises a peptide of sequence SEQ ID NO:1 or a (typical functional) variant thereof (i.e., a peptide having at least 90% identity with SEQ ID NO:1), and a peptide of sequence SEQ ID NO:2 or a (typical functional) variant thereof (i.e., a peptide having at least 90% identity with SEQ ID NO:2), wherein the (at least two) peptides are not connected to each other.

[0133] The group of two or more peptides described herein is advantageously formulated so that the peptides can be used alone or in combination. Therefore, each peptide in the group can be prepared in a separate form or in the form of a composition comprising a mixture of at least two peptides in the group. The composition can be a dietary / dietary composition or a pharmaceutical composition and can be used for treatment or prevention methods described herein.

[0134] In a specific aspect, the present invention relates to a composition comprising: at least two peptides or variants thereof as described herein that are not linked to each other, typically a group of (at least two) peptides that are not linked to each other, a nucleic acid or a group of nucleic acids encoding (at least two) peptides that are not linked to each other, an expression cassette or a group of expression cassettes allowing the expression of (at least two) peptides that are not linked to each other, a vector or a group of vectors allowing the expression of (at least two) peptides that are not linked to each other, or a cell or a group of cells allowing the expression of (at least two) peptides that are not linked to each other, and preferably comprises an acceptable support, carrier, excipient or vehicle as described herein, in particular a dietary and / or pharmaceutically acceptable support, carrier, excipient or vehicle.

[0135] The term "dietarily acceptable support" relates to a carrier that allows the subject to ingest and digest without risk a composition comprising a peptide or group of peptides, a nucleic acid sequence or group of nucleic acid sequences, an expression cassette or group of expression cassettes, a vector or group of vectors, or a cell or group of cells as described herein, and that is capable of protecting the peptides from any insults, in particular insults associated with the digestion of food, which could alter the peptides before they can produce a therapeutic effect at the right site and at the right time, depending on the nature and localization of the pain.

[0136] In certain aspects where the composition comprises two peptides, the first peptide and the second peptide may be present in the composition of the invention in equal or different proportions. In certain compositions, the first peptide and the second peptide are present in the composition in equal (equimolar) proportions, i.e., each peptide represents 50% of the peptide component of the composition, which means that the composition contains the same molar amount of each of the two peptides.

[0137] Another specific peptide composition may contain a specific peptide or multiple specific peptides in a greater proportion relative to another peptide or other peptides. For example, a specific peptide may represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the peptide component of the composition.

[0138] In the case of oral administration, the composition may further comprise at least one gastrointestinal protective agent, preferably an acid inhibitor, present in an amount effective to increase the stomach pH of the subject to at least 2, at least 3, at least 4, and more preferably to at least 5 or 6. The term "acid inhibitor" refers to a drug that inhibits gastric acid secretion and increases the pH of gastric fluid. Acid inhibitors may include, but are not limited to, H 2 Blockers, including cimetidine, ranitidine, bromotidine, palbutidine, lafutidine, lotidine, famotidine; proton pump inhibitors, including omeprazole, esomeprazole, pantoprazole, lansoprazole, dexlansoprazole, rabeprazole, paliprazole, leminoprazole and tenatoprazole; or any combination thereof.

[0139] "Pharmaceutically acceptable support / vehicle / carrier" can be a diluent, adjuvant or excipient used together with an active agent (i.e., at least two peptides of the present invention or variants thereof and optionally any other different active agents). Such a pharmaceutical carrier can be a sterile liquid, such as water or oil, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions, aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc.

[0140] The pharmaceutically acceptable support / vehicle / carrier may consist of or comprise ascorbic acid and / or an ascorbate.Examples of ascorbates are magnesium ascorbate, sodium ascorbate, calcium ascorbate and ascorbyl palmitate.

[0141] In one embodiment, the composition of the invention comprises ascorbic acid or an ascorbate salt.

[0142] When the composition, such as a pharmaceutical composition, is for oral administration, tablets or capsules can be prepared by conventional methods with pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by any method well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be present as dry products and reconstituted with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional methods with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl paraben or sorbic acid). The preparations may also contain buffer salts, flavoring agents, coloring agents and sweeteners as appropriate.

[0143] The composition of the present invention, such as a medicament, a preventive or therapeutic composition, may further comprise at least one additional (different) active compound. Preferably, the additional active compound is an active agent that is effective against pain. "Effective against pain" means that the active agent has analgesic or analgesic properties (which the subject can sense measurably). More preferably, the additional active compound is a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID) or an opioid.

[0144] SAIDs may include, but are not limited to, hydrocortisone, cortisone, ethxamethasone, prednisone, prednisolone, triamcinolone, dexamethasone, fludrocortisone, or any combination thereof.

[0145] NSAIDs may include, but are not limited to, celecoxib, rofecoxib, lumiracoxib, valdecoxib, parecoxib, etoricoxib, CS-502, JTE-522, L-745,337, NS398, aspirin, acetaminophen (considered an NSAID for purposes of this disclosure), ibuprofen, flurbiprofen, ketoprofen, naproxen, oxaprozin, etodolac, indomethacin, ketorolac, lornoxicam, meloxicam, piroxicam, droxicam, tenoxicam, nabumetone, diclofenac, meclofenamic acid, mefenamic acid, diflunisal, sulindac, tolmetin, fenoprofen, suprofen, benoxaprofen, aceclofenac, tolfenamic acid, oxyphenbutazone, azapropazone, phenylbutazone, or any combination thereof.

[0146] Opioids may include, but are not limited to, (dextro)propoxyphene, A-methylfentanyl, alfentanil, allylprodine, bezitamide, buprenorphine, butorphanol, carfentanil, norprodine, dextromoramide, dezocine, diacetylmorphine, dihydrocodeinone, dihydroetorphine, dimorphone, diphenoxylate, dipipanone, etorphine, fentanyl, ketobemidone, lefetamine, levoacetylmethadol, levomethorphan, Levomorphinol, loperamide, pethidine, meptazinol, methadone, methylmorphine, morphine, nalbuphine, nicomorphine, ormefentanil, olipaven, oxycodone, oxymorphone, PEPAP, paramorphine, pentazocine, phenazocine, piperidinamide, rhodine, remifentanil, sufentanil, tapentadol, tilidine, tramadol, or an opioid antagonist such as nalmefene, naloxone, naltrexone, or any combination thereof.

[0147] Therapeutic and preventive uses

[0148] The present specification discloses peptides, nucleic acids, expression cassettes, vectors, cells and groups thereof or any combination thereof, as well as compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells and / or groups thereof, as described above, which are used as active preventive or therapeutic ingredients, preferably as pharmaceutical compositions, drugs or medicines.

[0149] In a specific aspect, the peptides, in particular the isolated or synthetic peptide of SEQ ID NO: 1 and / or the isolated or synthetic peptide of SEQ ID NO: 2, preferably a group of said peptides, any group allowing the expression of such peptides, or any composition comprising such peptides, nucleic acids, expression cassettes, vectors, cells and / or groups as described herein, are used as pharmaceutical compositions, drugs or medicines, in particular for the prevention or treatment of pain.

[0150] In another aspect, the description relates to a peptide, a nucleic acid, an expression cassette, a vector or a cell as described herein and / or a panel as described herein for use as an active ingredient / agent for preventing or treating pain in a subject in need thereof.

[0151] The present description relates in particular to a product disclosed herein or a group of any product disclosed herein, such as a group of cells described herein, generally to a group of peptides or cells comprising or expressing such a group of peptides, for use [as an active ingredient] for preventing or treating pain in a subject in need thereof.

[0152] In a specific aspect, the inventors describe herein any product of the invention as described herein, such as a peptide, nucleic acid, expression cassette, vector, cell, and corresponding group thereof as described herein, or any combination thereof as described herein, for use in the preparation of a pharmaceutical composition, a medicament, or a drug for preventing or treating pain in a subject in need thereof. Also described herein are corresponding methods for preventing or treating pain, which generally include the step of administering the peptide, nucleic acid, expression cassette, vector, cell, group, and / or composition described herein to a subject in need thereof.

[0153] The present description particularly provides a method for preventing or treating pain in a subject in need thereof, comprising the step of administering a (therapeutically) effective amount of a product as described herein, such as, in particular, a group and / or composition as described herein, preferably a group of peptides comprising: a peptide of SEQ ID NO: 1 or a variant thereof having at least 90% identity with SEQ ID NO: 1, and a peptide of SEQ ID NO: 2 or a variant thereof having at least 90% identity with SEQ ID NO: 2, or a composition comprising such a group.

[0154] In a specific aspect, the inventors describe herein the use of any product of the present invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell as described herein, and their corresponding groups or any combination thereof, or a composition as described herein, for preventing or treating pain, in particular acute, subacute or chronic pain, preferably chronic pain.

[0155] Preferably, the pain is neuropathic pain (eg chemotherapy-induced peripheral neuropathic pain or chemotherapy-induced neuropathic pain), postoperative pain, inflammatory pain, hyperalgesia or allodynia.

[0156] In a specific aspect, the inventors describe herein the use of any product of the invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell as described herein and / or a group thereof as described herein, or any combination thereof or a composition as described herein, for preventing or treating neuropathic pain (such as chemotherapy-induced peripheral neuropathic pain or chemotherapy-induced neuropathic pain), postoperative pain or inflammatory pain.

[0157] In a specific aspect, the inventors describe herein the use of any product of the present invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell and their corresponding groups as described herein, or any composition thereof as described herein, for preventing or treating chronic neuropathic pain (such as chronic chemotherapy-induced peripheral neuropathic pain or chronic chemotherapy-induced neuropathic pain), chronic postoperative pain or chronic inflammatory pain.

[0158] In a more specific aspect, the inventors describe herein the use of any product of the invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell as described herein, and their corresponding groups, or any composition thereof as described herein, for preventing or treating osteoarthritis pain.

[0159] In a specific aspect, the inventors describe herein the use of any product of the invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell and their corresponding groups as described herein, or any composition thereof as described herein, for preventing or treating hyperalgesia, in particular thermal (such as heat or cold hyperalgesia, preferably cold hyperalgesia) or mechanical hyperalgesia, preferably mechanical hyperalgesia, even more preferably injury-induced mechanical hyperalgesia.

[0160] In another specific aspect, the inventors describe herein any product of the invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell and a corresponding group thereof as described herein, or any composition thereof as described herein, for use in preventing or treating allodynia, in particular mechanical allodynia. Preferably, the mechanical allodynia is mechanical allodynia caused by nerve damage or static mechanical allodynia.

[0161] In a further specific aspect, the inventors describe herein the use of any product of the invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell and their corresponding groups as described herein, or any composition thereof as described herein, for preventing or treating allodynia, in particular thermal allodynia, such as thermal allodynia (i.e., pain perceived to normally painless warm stimuli) or cold allodynia (i.e., pain perceived to normally painless cold stimuli), preferably cold allodynia.

[0162] In a specific aspect, the inventors describe herein the use of any product of the present invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell and their corresponding groups as described herein, or any composition thereof as described herein, for preventing or treating mechanical hypersensitivity (also known as hypersensitivity to mechanical stimulation or simply hyperalgesia), preferably mechanical hypersensitivity caused by injury.

[0163] In a specific aspect, the inventors describe herein any product of the invention as described herein, such as a peptide, a nucleic acid, an expression cassette, a vector, a cell as described herein, and their corresponding groups, or any composition thereof as described herein, for use in preventing or treating pain caused by sunburn or pain caused by ice burns.

[0164] Treatment can improve one or more sensations such as touch, burning or cold, pins and needles, numbness, itching, severe pain, and difficulty correctly perceiving temperature. In a specific aspect, the treatment can eliminate pain. In another specific aspect, the treatment reduces the symptoms of pain, particularly the symptoms of neuropathic pain (allodynia and / or hyperalgesia). The methods of the present invention can make neuropathic pain more manageable (i.e., improve quality of life), even if they do not eliminate neuropathic pain.

[0165] Pain, in particular (chronic) neuropathic pain, can be assessed using standard tests well known to those skilled in the art, whether it has been treated using a specific peptide of the invention. For example, the assessment of pain sensitivity in a subject has been standardized using quantitative sensory tests (pinprick test, pressure analgesia, von Frey pain test, touch, pinching or light pressure with fingers) or using a pain rating scale.

[0166] Subjects

[0167] In the context of the present invention, the subject or patient is an animal, preferably a mammal. In a specific aspect, the subject is a domestic animal, such as a horse, dog, cat, cow, etc. In another specific and preferred aspect, the subject is a human.

[0168] A subject suffering from chronic pain originating from neuropathic pain may suffer from a disease typically associated with such neuropathic pain, such as fibromyalgia, complex regional pain syndrome, postherpetic neuralgia, Ehlers-Danlos syndrome, and erythromelalgia.

[0169] In certain aspects, the subject suffers from fibromyalgia (FM). Fibromyalgia is a syndrome characterized by chronic musculoskeletal pain (Siracusa et al., 2021). FM is caused by a phenomenon of central sensitization, which is characterized by dysfunction of neural circuits involved in the perception, transmission and treatment of afferent noxious stimuli, which generally manifests as pain at the level of the motor system. The main symptoms of the disease are muscle stiffness, joint stiffness, insomnia, fatigue, mood disorders, cognitive dysfunction, anxiety, depression, systemic sensitivity and inability to carry out normal daily activities. FM may also be associated with specific diseases, such as infections, diabetes, rheumatic diseases and / or psychiatric or neurological diseases.

[0170] In a specific aspect, the inventors describe herein a peptide, a nucleic acid, an expression cassette, a vector, a cell as described herein, and a corresponding group thereof (i.e., a group of such peptides, nucleic acids, expression cassettes, vectors or cells) or a composition as described herein comprising such a peptide, nucleic acid, expression cassette, vector, cell or group for use in preventing or treating fibromyalgia (FM).

[0171] In another aspect, the subject suffers from complex regional pain syndrome (CRPS). Complex regional pain syndrome is a chronic neuropathic disorder involving the extremities characterized by severe pain with sensory, autonomic, motor and trophic disturbances (Goh et al., 2017). The condition may be caused by surgery, trauma or minor injuries and has a variable course, ranging from mild and self-limited, to a chronic condition that affects daily living activities and health-related quality of life. CRPS can be divided into two types: CRPS types I and II are characterized by the absence or presence of identifiable nerve damage. CRPS type I is a syndrome that usually develops after an initiating noxious event, is not confined to the distribution of a single peripheral nerve, and is out of proportion to the precipitating event. It is associated with edema, changes in skin blood flow, abnormal sweat gland activity in the painful area, allodynia and hyperalgesia, and usually involves the distal end of the affected limb or has a distal to proximal gradient. CRPS type II can be defined as burning pain, allodynia and hyperalgesia that occur in an area of ​​the limb after a localized injury to a nerve or one of the major branches innervating the area.

[0172] In a specific aspect, the inventors describe herein the use of a peptide, nucleic acid, expression cassette, vector, cell as described herein and its corresponding groups (i.e., groups of such peptides, nucleic acids, expression cassettes, vectors or cells) or a composition as described herein comprising such a peptide, nucleic acid, expression cassette, vector, cell or group for preventing or treating Complex Regional Pain Syndrome (CRPS).

[0173] On the other hand, the subjects suffered from postherpetic neuralgia (PHN). Postherpetic neuralgia is the most common complication of herpes zoster (HZ), an infection caused by the reactivation of dormant varicella zoster virus in sensory ganglia (Ngo et al., 2020). It is characterized by a localized blistering rash with associated dermatomal pain. PHN is defined as pain that persists for at least 90 days after the first appearance of the HZ rash, and it significantly reduces the quality of life of affected patients. PHN is subdivided into the irritable nociceptor model and the deafferentation model. During VZV reactivation, the virus replicates and spreads from the spinal ganglia to their respective peripheral environment. This proliferation induces an immune response and inflammation that damages the peripheral nerves. This damage reduces the ability of neurons to inhibit pain, thereby lowering the threshold for depolarization of pain signals. This leads to the perception of pain in response to non-painful stimuli, a process called peripheral sensitization. Repetitive activation of C-subtype nociceptors also leads to an enhanced dorsal horn excitatory state. Direct damage caused by HZ weakens the descending inhibitory pain pathway, resulting in chronic activation of second-order neurons in the dorsal horn. Furthermore, HZ patients with PHN have been reported to have a loss of inhibitory gamma-aminobutyric acid (GABA)-producing interneurons in the dorsal horn compared to HZ patients without PHN. These factors amplify all subsequent responses to afferent inputs in a process known as central sensitization. In PHN, this process is accompanied by an anatomical reorganization of low-threshold mechanoreceptive afferents (known as Aβ fibers) that normally transmit innocuous touch stimuli to the central nervous system. When viral insults result in the loss of peripheral C-nociceptors, these fibers connect in a compensatory manner to the second-order neurons that were originally connected to the C-nociceptor afferents. This process is known as deafferentation, and patients with allodynia exhibit a profound loss of sensory function.

[0174] In a specific aspect, the inventors describe herein the use of a peptide, nucleic acid, expression cassette, vector, cell as described herein and its corresponding groups (i.e., groups of such peptides, nucleic acids, expression cassettes, vectors or cells) or a composition as described herein comprising such peptide, nucleic acid, expression cassette, vector, cell or group for preventing or treating postherpetic neuralgia (PHN).

[0175] In another aspect, the subject suffers from erythromelalgia (EM). Erythromelalgia is a rare episodic acromegaly syndrome that primarily affects the lower extremities bilaterally symmetrically or unilaterally, with the classic triad of erythema, warmth, and burning pain (Maria Bibiana Leroux, 2018). EM is grouped with chronic pain syndromes. Primary EM is an autosomal dominant inherited disease, coded by OMIN (Online Mendelian Inheritance in Man) as #133020. It is associated with alterations in the alpha subunit protein of the type 9 sodium channel (SCN9A), affecting the Nav1.7 channel that is primarily expressed in dorsal root ganglia and sympathetic ganglion neurons. Secondary EM is associated with myeloproliferative disorders, paraneoplastic, autoimmune diseases, exposure to toxins, and infections.

[0176] In a specific aspect, the inventors describe herein a peptide, a nucleic acid, an expression cassette, a vector, a cell as described herein, and a corresponding group thereof (i.e., a group of such peptides, nucleic acids, expression cassettes, vectors or cells) or a composition as described herein comprising such a peptide, nucleic acid, expression cassette, vector, cell or group for use in preventing or treating erythromelalgia (EM).

[0177] In a specific aspect, the inventors describe herein the use of peptides, nucleic acids, expression cassettes, vectors, cells, and their corresponding groups (i.e., groups of such peptides, nucleic acids, expression cassettes, vectors or cells) as described herein, or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells or groups as described herein, for preventing or treating any pain-related disorder in which signals transmitted by nociceptors or interneurons are impaired.

[0178] The inventors previously determined that Myo1a, when expressed in combination with + / + The absence, reduced or insufficient expression or non-functional expression of myosin IA (Myo1a) in a subject makes the subject susceptible to injury-induced chronic mechanical pain and / or inflammation-induced chronic thermal pain when compared to the expression observed in a reference subject (WO2017153424).

[0179] In another aspect, the subject is a subject having one or two mutated alleles in the myo1A gene. A "mutated" allele refers to a substitution, deletion, or insertion of the myo1A gene nucleic acid (in a coding or non-coding region) that alters the expression or expression level of Myo1a. The mutation may affect one or more nucleobases. The substitution may be a single nucleotide polymorphism (SNP).

[0180] In a specific aspect, the inventors describe herein the use of peptides, nucleic acids, expression cassettes, vectors, cells and their corresponding groups (i.e., groups of such peptides, nucleic acids, expression cassettes, vectors or cells) as described herein, or compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells or groups as described herein, for preventing or treating chronic mechanical pain caused by injury and / or chronic thermal pain caused by inflammation in such subjects.

[0181] dose

[0182] The product according to the present invention, in particular a peptide, a group of peptides or a composition comprising such a group or allowing its expression, is preferably directly administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of the peptide of interest required to treat, improve or prevent pain in a subject, in particular a peptide of SEQ ID NO:1 and SEQ ID NO:2 or a functional variant thereof. The therapeutically effective amount can be preliminarily estimated in a cell culture assay or an animal model. An animal model can also be used to determine the appropriate concentration range and route of administration of the peptide (group of peptides). This information can be used to determine useful doses and routes for human administration.

[0183] The dosage of the peptide used in the methods described herein may vary depending on the general health, age, sex and weight of the subject, the nature and severity / intensity of the pain, the time, frequency and duration of administration, the specific peptide used, the drug combination, reaction sensitivity and tolerance / response to treatment. The appropriate effective dosage can be determined by routine experimentation and is at the discretion of the clinician.

[0184] The dosage can be administered in pill form or divided into several portions ( / units) and administered separately throughout the day. In other words, the treatment can be a single dose regimen or a multiple dose regimen. The effective dose can be administered continuously for several days, weeks, months or years.

[0185] In order to obtain a suitable analgesic or analgesic effect, the effective total dose of the peptides or variants thereof present in the group of peptides described herein by the inventors, or in a composition comprising such a group of peptides (generally both the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2) is 1 μg to 10g per unit dose, preferably 1 μg to 100 mg, more preferably 25 μg to 10 mg, and even more preferably 2 mg to 10 mg.

[0186] In a specific aspect of the invention, the group of peptides or a composition comprising such a group of peptides is administered to a mammal at a dose of 1 μg / kg body weight (i.e., 1 μg / kg / day) to 100 mg / kg / day, preferably 2.5 μg / kg / day to 0.6 mg / kg / day every 24 hours.

[0187] When the subject is a human, the effective dose of the peptide or variant thereof described herein by the inventors administered to the subject (if the subject is a human subject) is preferably between 2.5 μg / kg / day and 0.6 mg / kg / day, preferably between 5 μg or 10 μg / kg / day and 0.5 mg / kg / day, and more preferably between 50 μg or 75 μg / kg / day and 0.3 mg / kg / day.

[0188] In a specific aspect of the invention, the set of peptides or a composition comprising the set of peptides is administered at a dosage wherein the peptide of SEQ ID NO: 1 and the peptide of SEQ ID NO: 2 are at an equivalent molar concentration.

[0189] In a more specific aspect of the invention, the group of peptides or a composition comprising such a group of peptides is administered at a dose comprising 50 to 150 μg / kg of the peptide of SEQ ID NO: 1 and 100 to 250 μg / kg of the peptide of SEQ ID NO:2.

[0190] Delivery of a product as described herein, in particular a peptide or composition as described herein, to a subject in need thereof can be achieved by any route.

[0191] The products described herein can be administered, for example, intramuscularly, intravenously, intraperitoneally, orally (perorally), anally, cutaneously, subcutaneously, topically, dermally, transdermally or intrathecally. Preferably, the products described herein are administered subcutaneously, orally or intravenously, even more preferably subcutaneously or orally.

[0192] As shown in the experimental part, the inventors have demonstrated that a group of peptides comprising a peptide of SEQ ID NO: 1 and a peptide of SEQ ID NO: 2 has an analgesic effect when administered subcutaneously to treat pain, in particular neuropathic pain. Surprisingly, an analgesic effect was also observed when a group of peptides comprising a peptide of SEQ ID NO: 1 and a peptide of SEQ ID NO: 2 was administered orally (orally) or intravenously. Even more surprisingly and advantageously, the group of peptides of the present invention, when administered orally or subcutaneously, showed a better analgesic effect than the TAFA-4 (full length) protein, which makes it more convenient and suitable for administration to patients than the full length protein.

[0193] Advantageously, the set of two peptides of the invention or a composition comprising such a set, although easier to produce (and therefore cheaper) than the full-length TAFA-4 protein, induces a higher analgesic activity / effect when administered subcutaneously than the latter. Even more surprisingly, when administered orally (per oral), the set of peptides of the invention exhibits a better analgesic effect than the TAFA-4 (full-length) protein, which makes it more convenient and suitable for administration to patients than said TAFA-4 full-length protein. Intravenous injection of the set of two peptides advantageously provides a third route of administration for the treatment of neuropathic pain.

[0194] Reagent test kit

[0195] Each product described herein, such as each product described herein, preferably each peptide described herein, each group of peptides described herein or each composition described herein, may be part of a kit.

[0196] Typical kits of the invention comprise at least two products described herein, selected from a peptide or variant thereof, a nucleic acid, an expression cassette, a vector, a cell, a group of peptides, a group of nucleic acids, a group of expression cassettes, a group of vectors, a group of cells, and compositions comprising such peptides, nucleic acids, expression cassettes, vectors, cells or groups, and optionally at least one additional compound of different activity effective against pain. In a specific aspect, the kit further comprises written instructions for using the kit.

[0197] The kit is preferably a kit of parts comprising at least two parts (e.g., two different containers), wherein the first part comprises a peptide, a nucleic acid, an expression cassette, a vector, a cell, a group of peptides, a group of nucleic acids, a group of expression cassettes, a group of vectors or a group of cells, or a composition disclosed herein, and the second part comprises at least one additional compound with different activity effective against pain. Preferably, the active compound effective against pain is a steroidal anti-inflammatory drug (SAID), a non-steroidal anti-inflammatory drug (NSAID) or an opioid disclosed herein.

[0198] In a specific aspect, the kit comprises i) a first peptide or a nucleic acid sequence encoding such a peptide, the first peptide being SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1, ii) a second peptide or a nucleic acid encoding such a peptide, the second peptide being a second peptide of SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2, the first peptide and the second peptide or the nucleic acid encoding the peptide being in different containers, and optionally, iii) at least one additional compound of different activity effective against pain, and / or iv) written instructions for using the kit.

[0199] In one aspect, the peptides, nucleic acids, expression cassettes, vectors, cells, groups or compositions in the kit are in a form suitable for intramuscular, intravenous, intraperitoneal, oral (peroral), anal, cutaneous, subcutaneous, topical, dermal, transdermal or intrathecal routes, preferably subcutaneous or oral routes, even more preferably oral routes.

[0200] In another aspect, at least one additional different active compound effective against pain is in a form suitable for intramuscular, intravenous, intraperitoneal, oral (per os), anal, cutaneous, subcutaneous, topical, dermal, transdermal or intrathecal routes.

[0201] Depending on the nature, origin and intensity of the pain to be treated, and also on the nature of the contents of the peptide, nucleic acid, expression cassette, vector, cell, group or composition and on the nature of the at least one additional different active compound effective against pain, the products can be administered together or not together, simultaneously / concomitantly or sequentially.

[0202] The present invention also relates to the use of the kit of the present invention in vivo, ex vivo or in vitro for preventing or treating pain as described above (e.g. chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia or allodynia). The kit of the present invention can also be used to prevent or treat acute or subacute pain.

[0203] The present invention also discloses the use of the kit of the present invention in preparing a pharmaceutical composition, a drug or a medicine for preventing or treating the above-mentioned pain in a subject in need thereof, such as chronic pain, neuropathic pain, postoperative pain, inflammatory pain, hyperalgesia or allodynia. The kit of the present invention can also be used to prepare a pharmaceutical composition, a drug or a medicine for preventing or treating acute or subacute pain in a subject in need thereof.

[0204] Research Tools

[0205] In certain aspects, the products of the invention may also be used in a research setting.

[0206] The present invention also encompasses the use of a peptide as described herein or a variant thereof, in particular a group of peptides or any other product as described herein above, as a research tool.

[0207] Nucleic acids encoding the peptides or variants thereof described herein, expression cassettes or vectors allowing their expression, or cells comprising such nucleic acids or modified using such expression cassettes or vectors are typically used to express or regulate the expression of the peptide of interest in vitro or ex vivo, in particular the peptide of SEQ ID NO: 1 or a functional variant thereof having at least 90% identity with SEQ ID NO: 1 and / or the peptide of SEQ ID NO: 2 or a functional variant thereof having at least 90% identity with SEQ ID NO: 2.

[0208] These tools can be used to study pain for modulation of neuronal excitability in biological tissues or cell cultures, for example to study mechanically and / or chemically induced nociceptive signaling.

[0209] The present invention also relates to the use of nucleic acids encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1 and a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2, or an expression cassette or vector allowing the expression thereof, to express or regulate the expression (level) of a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1 and a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2 in biological tissues or cell cultures.

[0210] The nucleic acid molecules described herein can also be used to produce transgenic animals. This can be achieved by local somatic cell modification or by germline treatment so that the germ cells contain heritable modifications. Therefore, the present invention also relates to transgenic organisms (e.g., animals) comprising (i.e., containing) nucleic acids of SEQ ID NO:3 and / or SEQ ID NO:4 or variants thereof; expression cassettes or vectors comprising nucleic acids of SEQ ID NO:3 and / or SEQ ID NO:4 or variants thereof; or expressing a peptide of SEQ ID NO:1 or a peptide having at least 90% identity to SEQ ID NO:1 and / or a peptide of SEQ ID NO:2 or a peptide having at least 90% identity to SEQ ID NO:2. Therefore, the present invention also relates to host cells or transgenic organisms, in particular transgenic animals, containing a nucleic acid sequence encoding any (at least two) groups of peptides described herein or an expression cassette or vector allowing their expression.

[0211] The following examples are provided to demonstrate and further illustrate certain preferred aspects of the present invention, but should not be construed as limiting the scope of the present invention. Example

[0212] Materials and methods

[0213] Peptide synthesis

[0214] All peptides were synthesized using Fmoc-SPPS on a Symphony X instrument (Protein Technologies, Inc., USA) at a 200 μmol scale. Synthetic grade Fmoc-AA reagent was purchased from Iris Biotech. Specific L-pseudoproline dipeptide was used to facilitate peptide synthesis. HATU, 2-chlorotrityl chloride resin (200-400 mesh, 1.2 mmol / g), RINK amide resin (200-400 mesh, 0.42 mmol / g) were purchased from IRIS Biotech. DMF, DCM, methanol, ether and HPLC grade acetonitrile were purchased from Aldrich. The standard deprotection-coupling cycle for each residue consisted of six steps:

[0215] 1. Wash with 5 mL DMF (3x 30 sec)

[0216] 2. Deprotect the Fmoc group with 5 mL of 20% piperidine in DMF (3 x 3 min)

[0217] 3. Wash with 5 mL DMF (3x 30 sec)

[0218] 4. Coupling of Fmoc-AA (2x 60 min)

[0219] a. Dissolve 5 ml Fmoc-AA at 200 mM in DMF

[0220] b. Dissolve 2 ml HATU at 500 mM in NMP

[0221] c. 2ml iPr 2 NEt was dissolved in NMP at 1M

[0222] 5. Use 5mL 10% Ac 2 O capping in DMF (7 min)

[0223] 6. Wash with 5 mL DMF (3x 30 sec)

[0224] After the synthesis was complete, the peptide resin was washed 3 times with DMF and 3 times with DCM. The peptide resin was then cleaved with one of three TFA cleavage mixtures (per 100 μmol scale synthesis):

[0225] o 35mL TFA / 1000μL TIS / 2000μL water, 1000mg DTT

[0226] After 3 h of TFA cleavage, the cleavage solution was filtered from the resin and the peptide was precipitated by addition to 40 mL of ice-cold ether. After >1 h at -20 °C, the ether solution was centrifuged at 3500 RCF and the supernatant was decanted. The precipitate was washed twice more with ether and then dried in a vacuum desiccator for >3 h before solubilization, analytical characterization and purification.

[0227] General Method for Preparing Peptide Resin Hydrazide

[0228] 0.35 mmol Fmoc-NH-NH 2 Dissolve in 30 mL DCM and then add 1.5 mmol DIPEA to the solution. The solution is then added to 1 g of resin and mixed for 2 h at room temperature. Unreacted groups are capped with 17:2:1 DCM:MeOH:DIPEA for 30 min. The Fmoc-AA-resin is then washed 3 times with DMF, 3 times with DCM, and dried under vacuum for >20 min. Loading is determined by titrating the Fmoc group deprotection (UV, 301 nm).

[0229] Analysis and purification

[0230] The peptides were analyzed by UPLC and ESI-MS mass spectrometry. The instrument was equipped with BEH C18 (WATERS), 150*2.1 mm (150×2.1 mm) (flow rate: 0.6 mL / min). Solvents A and B were 0.1% TFA in water and 0.1% TFA in acetonitrile.

[0231] A reverse phase column (Vydac Denali prep C-18, 10 μm, The crude peptide was purified by preparative reverse phase HPLC (Waters DeltaPrep 4000) using a 50×300 mm column and an appropriate gradient of increasing concentrations of buffer B in buffer A (flow rate of 80 mL / min). Fractions containing the purified target peptide were identified by UV measurement at 214 nm (Waters 2489 UV / visible detector), and the selected fractions were then pooled and lyophilized.

[0232] Synthesis of Ac-CFPGQVAGTTRAQPSCVEASIVIQKWW-NHNH 2 (Peptide F1 - SEQ ID NO:1):

[0233] Peptide F1 (Ac-CFPGQVAGTTRAQPSCVEASIVIQKWW-NHNH) was prepared by standard Fmoc-SPPS as described above using hydrazide resin. 2 ), followed by standard peptide cleavage conditions (described above).

[0234] After standard cleavage procedures, the crude F1 peptide was dissolved in water and purified on RP-HPLC. Fractions containing the purified target peptide were identified and selected fractions were pooled and lyophilized.

[0235] Synthesis CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR-NH 2 (Peptide F2 - SEQ ID NO: 2):

[0236] Peptide F2 (CHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR-NH 2 ).

[0237] After standard cleavage procedures, the crude F2 peptide was dissolved in water and purified on RP-HPLC. Fractions containing the purified target peptide were identified and selected fractions were pooled and lyophilized.

[0238] Mouse

[0239] C57 / Bl6J mice (8 to 12 weeks old) were purchased from Charles River Laboratories. All experiments used male and female mice. As no differences were found between males and females, the data from both sexes were combined. Mice were housed under standard housing conditions (22°C, 40% humidity, 12 h light cycle, free access to food and water). We made special efforts to minimize the number of mice used in this study, as well as the stress and suffering they were subjected to. All experiments were performed in accordance with the European Guide for the Care and Use of Laboratory Animals (Council Directive 86 / 609 / EEC). All experimental procedures were approved by the Independent Ethics Committee for Animal Experimentation (APAFIS), in compliance with the requirements of French law and in accordance with the relevant institutional provisions of the French legislation on animal experiments. All experiments were performed in accordance with the ARRIVE guidelines.

[0240] Pain Model 1: Sparing Nerve Injury (SNI) Model - Neuropathic Pain.

[0241] The Sparing Nerve Injury (SNI) model developed by Decosterd and Woolf, 2000; Pain, Vol. 87, p 149-158 was used; it is used as a model of neuropathic pain. The SNI model involves transection of the common peroneal nerve of the tibial branch and the sciatic nerve: the sural nerve remains intact. The latter subsequently develops symptoms of neuropathic pain, accompanied by severe mechanical allodynia. The SNI model has many advantages:

[0242] - Neuropathic pain is persistent. This allowed us to capture the habituation phenomenon after repeated injections of the peptide.

[0243] -The pain produced is intense.

[0244] -The model is highly repeatable.

[0245] Mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP), and the left sciatic nerve was exposed under sterile conditions. The distal trifurcation of the sciatic nerve was identified, and the tibial and common peroneal branches were ligated with polypropylene non-absorbable 6-0 sutures (Ethicon); 1 mm was resected, leaving the sural branch intact. The wound was closed with sutures, and the animals were allowed to recover and returned to their cages.

[0246] Pain model 2: Paw incision - postoperative pain.

[0247] Paw incision surgery was performed as described by Brennan and colleagues (1999) (Brennan, 1999). Mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP), and a longitudinal incision was made along the skin and fascia of the right hind paw. The flexor digitorum brevis muscle was lifted longitudinally using forceps and a scalpel was used to cut the muscle in half. The wound was closed with sutures and the animals were allowed to recover and returned to their cages. Paw incision was used as a postoperative pain model.

[0248] Pain model 3: Carrageenan injection - inflammatory pain.

[0249] The inventors injected 20 μl of 1% λ-carrageenan (Sigma-Aldrich, 22049-5G-F) in 1x PBS into the plantar surface of the left hind paw of mice using a Hamilton syringe.Carrageenan injection was used as an inflammatory pain model.

[0250] Pain Model 4: Chronic Constriction Injury (CCI) Model - Neuropathic

[0251] CCI neuropathic pain model: Chronic constriction injury (CCI) was performed as previously described by Bennett and Xie 1988 (Aperipheral mononeuropathy in rats that produces disorders of pain sensationlike those in man. Pain vol 33: pp87-107). Briefly, mice were anesthetized with ketamine / xylasine (100 mg / kg and 10 mg / kg ip, respectively) and two ligatures (6-0 Monocryl, Ethicon) were loosely tied around the common sciatic nerve (about 1 mm apart) to induce unilateral peripheral mononeuropathy. The nerve was contracted to an almost imperceptible degree, so the circulation of the extraneural vascular system was not interrupted.

[0252] Pain model 5: capsaicin inflammatory pain.

[0253] Capsaicin (Sigma) stock solution (dissolved in 16.7 mg / mL absolute ethanol) was diluted in 0.9% NaCl at 0.25 mg / mL and 10 μL was injected into the plantar surface of the left hind paw of mice using a Hamilton syringe and a 26G needle.

[0254] pain Model 6: Osteoarthritis pain model (monoiodoacetate model).

[0255] The MIA model is a standard model of joint destruction in rodent osteoarthritis. Briefly, mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP), and then 1 mg / 10 μL MIA (monoiodoacetate, Sigma) in 0.9% NaCl was injected into the synovial fluid of the left hind paw knee of the mice using a Hamilton syringe and a 26G needle as described by Pitcher et al. (2016).

[0256] Pain model 7: UV burn pain model

[0257] Mice were anesthetized with ketamine (100 mg / kg IP) and xylazine (10 mg / kg IP) and completely covered, leaving only the plantar side of one hind paw. The paw was irradiated for 60 minutes at a distance of 20 cm using a UV lamp with a wavelength of 312 nm (VL-215.MC Vilmer). Once awake, the animal was returned to its cage.

[0258] Pain model 8: frostbite pain model

[0259] Mice were anesthetized with ketamine (100 mg / kg Ip) and xylazine (10 mg / kg IP). The hind paws were exposed to cold via a copper tube placed in dry ice and contacted by a NaCl-soaked filter paper piece for 40 seconds. Once awake, the animals were returned to their cages.

[0260] VonFrey Test

[0261] Von Frey uses Von Frey (VF) hairs or fibers (i.e., small segments of nylon rods, approximately 50 mm in length) to test the sensitivity of rodents to mechanical stimulation. In this test, the animal stands on an elevated mesh platform and Von Frey hairs are inserted through the mesh into the animal's hind paws. The animal's normal response includes retracting or licking or shaking the paw. The "up and down" Von Frey method is used to determine the mechanical force required to elicit a paw retraction response in 50% of the animals. Here, mice are placed in a plastic chamber on a wire mesh and stimulated with von Frey filaments (Vivotech) using the "up and down" method (45), starting with a 1 g filament and using 0.04 and 4 g filaments as cutoffs.

[0262] Statistical analysis

[0263] The results are expressed as mean ± SEM. Statistical analysis was performed using Prism 7 (Graphpad Software, La Jolla, CA, USA).

[0264] result

[0265] Example 1: Pain Model 1: Sparing Nerve Injury (SNI) Model - Neuropathic Pain.

[0266] 1.1 TAFA-4 full-length protein ("TAFA-4 FL") and two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 Comparison of the analgesic effect of the groups ("peptide 1+2") in case of subcutaneous administration.

[0267] The purpose of these experiments was to evaluate the analgesic effect of a group of two peptides according to the invention (the group identified herein as "Peptide 1+2" or "Peptide F1+F2") in the sparing nerve injury (SNI) model of neuropathic pain by subcutaneous injection and to compare this effect with that obtained with the full-length TAFA-4 protein ("TAFA-4FL").

[0268] The experiments were performed on eight-week-old male WT C57Bl6 mice. Four groups of mice, 6 to 12 mice each, were used. The peptide of SEQ ID NO:1 and the peptide of SEQ ID NO:2 were resuspended in 0.9% NaCl at [0.6 mg / mL]. Both peptides were diluted to obtain equivalent molar concentrations (12.8 μg / ml for the peptide of SEQ ID NO:1 and 17.2 μg / ml for the peptide of SEQ ID NO:2) so that they were used in this study at a final total concentration of 0.3 mg / kg of "peptide 1+2". Similarly, TAFA-4FL was resuspended in 0.9% NaCl, diluted and used at a final concentration of 0.3 mg / kg.

[0269] 0.9% NaCl solution was used as a negative control (vehicle), and 3 mg / kg of pregabalin (a well-known drug for the treatment of neuropathic pain) was used as a positive control.

[0270] The SNI model was established by measuring the baseline threshold ("baseline") of mice using the up / down method with Von Frey (VF) filaments [showing the change in mechanical force (weight equivalent in grams) required to elicit a paw withdrawal response in 50% of mice under different conditions over time]. Mice were anesthetized; the tibial and peroneal nerves were ligated; and then both nerves were cut. Neuropathy quickly developed in the intact sural nerve. Whether neuropathy developed was determined 3 days after surgery. A decrease in the threshold for the response to the Von Frey filaments of the ipsilateral paw was observed.

[0271] 14 days after surgery, the response threshold was measured again (see Figure 1 Then, the experimenters blindly injected 100 μl / 10 g of each solution containing the two peptide groups, TAFA-4 full-length protein, vehicle or pregabalin subcutaneously into the mice.

[0272] Response thresholds were measured 1 hour, 2 hours and then 4 hours after administration / injection.

[0273] At day 14 after SNI, all mice showed a dramatic decrease in mechanical thresholds compared with baseline thresholds, indicating a strong mechanical hypersensitivity (see Figure 1 ). Subcutaneous administration of the vehicle had no effect. Surprisingly, at 1 hour after administration, the analgesic effect of the group / combination of the two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("Peptide 1+2") was the greatest and significantly higher than the analgesic effect of the full-length protein of TAFA-4 ("TAFA4FL") (p value <0.01), and also higher than pregabalin. Statistical analysis showed that the results of the group / combination of the two peptides were highly significant at 1 hour and 2 hours compared to the negative control (vehicle) (p value <0.001).

[0274] 1.2 TAFA-4 full-length protein ("TAFA-4 FL") and two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 Comparison of the analgesic effects of the groups ("peptide 1+2") in the case of oral administration.

[0275] The protocol described in 1.1 was used, except that the administration of both peptide groups, TAFA-4FL, vehicle and pregabalin was completed 14 days after surgery using oral administration.

[0276] For oral administration, the two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were diluted at a concentration of 30 μg / mL in 1% w / v hydroxypropylmethylcellulose (Sigma-aldrich #423238, batch number MKCD3665), 0.5% v / v Tween 80 (Euromedex #2002-A, batch number 100412 / 16S407) solution, hereinafter referred to as HPMC solution or "vehicle". A similar protocol was applied to TAFA-4FL.

[0277] Vehicle, TAFA-4FL solution and solution containing both peptides were orally administered using two gavage needles. Pregabalin (3 mg / kg) was administered by oral gavage (oral) by an experimenter different from the one who performed the VF measurements so that the latter was blinded to the treatment (n=8 per treatment). Administration was performed 14 days after surgery.

[0278] On day 14 after SNI, all mice showed a sharp decrease in mechanical threshold ( Figure 2). Oral administration of the vector had no effect. The TAFA-4 full-length protein did not exhibit significant analgesic effects when administered orally (i.e., per mouth). Surprisingly, oral administration of the group / combination of the two peptides of the present invention induced a statistically significant reversal effect on the mechanical threshold immediately 1 hour after administration (p-value <0.01), with the maximum effect occurring 2 hours after administration (p-value <0.001). At 1 hour and 2 hours after administration, the analgesic effect of the group / combination of the two peptides of the present invention was similar to that of pregabalin, however, at 2 hours after administration, it was significantly higher than the analgesic effect of the TAFA-4 full-length protein ("TAFA4FL"). From Figure 2 It can be concluded that the group / combination of the two peptides of the present invention has a better analgesic effect than the full-length TAFA-4 protein when administered orally.

[0279] 1.3 Analgesic effect of a group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("peptide 1+2") administered intravenously

[0280] Use the protocol described in 1.1, except that administration of both peptide groups or vehicle is completed 14 days post-surgery using the intravenous route.

[0281] The peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were resuspended in 0.9% NaCl at a total concentration of 150 μg / mL.

[0282] As previously described, Von Frey filament measurements were performed using the up / down method to determine the baseline. Then, the SNI model was put in place. The reaction threshold was measured fourteen days after surgery (14 days-D14) to examine the occurrence of neuropathic pain. Then, a blind injection of 20 μL / 10g of a solution containing a group of two peptides at a total concentration of 300 μg / kg was slowly performed in the tail vein. The reaction threshold was measured 1 hour, 2 hours, and then 4 hours after intravenous administration. Vehicle (n=11) was also administered intravenously.

[0283] At day 14 after SNI, all mice showed a dramatic decrease in mechanical threshold compared with baseline thresholds, indicating a strong mechanical hypersensitivity ( Figure 3 ). Intravenous administration of the vehicle had no effect. Surprisingly, a strong and significant analgesic effect was observed for the group of two peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("Peptide 1+2"), with the maximum reversal effect on the mechanical threshold observed at 2 h (p<0.001).

[0284] In summary, the present experiment shows that the group of two peptides of the present invention induces analgesic effects in the SNI model (neuropathic pain model) by subcutaneous, oral and intravenous administration. Advantageously, when administered subcutaneously, the analgesic effect of the group of two peptides of the present invention at the very early stage (i.e., 1 h after administration) is more significant than the analgesic effect observed with the TAFA-4 (full length) protein. Even more surprisingly, the group of two peptides of the present invention, when administered orally, exhibits a better analgesic effect than the TAFA-4 (full length) protein at 2 h after administration.

[0285] The inventors' results demonstrated the analgesic effect of the two peptide groups of SEQ ID NO: 1 and SEQ ID NO: 2 in a neuropathic pain model. The mechanical allodynia (reduced pain sensitivity threshold) induced by this model can be reduced by subcutaneous, intravenous or oral administration of the peptide groups of the present invention.

[0286] Advantageously, the set of two peptides of the invention, or a composition comprising such a set, is not only easier to produce (and therefore cheaper) than the full-length TAFA-4 protein, but also induces a higher analgesic activity / effect when administered subcutaneously compared to the latter. Even more surprisingly, when administered orally (per oral), the set of peptides of the invention exhibits a better analgesic effect compared to the TAFA-4 (full-length) protein, which makes it more convenient and suitable for administration to patients than said TAFA-4 full-length protein. Intravenous injection of the set of two peptides advantageously provides a third route of administration for the treatment of neuropathic pain.

[0287] Example 2: Paw incision - postoperative pain -

[0288] The aim of these experiments was to evaluate the analgesic effect of a set of two peptides according to the invention, SEQ ID NO: 1 and SEQ ID NO: 2 (this set is identified herein as "Peptide 1+2"), in a postoperative pain model (Brennan paw incision model) by subcutaneous injection.

[0289] The experiment was conducted on eight-week-old male and female mice WT C57B16. 12 males and 12 females were used for the "peptide 1 + 2" treated group, while 11 males and 11 females were used for the vehicle group. The peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were resuspended in ultrapure water at [4 mg / mL]. In this study, both peptides were diluted to obtain a final total concentration of "peptide 1 + 2" of 0.3 mg / kg.

[0290] At different time points: one day before surgery (D-1), the day of surgery (D0), and D+1 and D+2 after surgery, "peptide 1+2" or vehicle was subcutaneously administered twice a day. The administration of D0 was performed 1 hour before surgery and 1 hour after surgery (and waking up). Mechanical threshold response measurements were performed on D+1 (before injection on D+1), D+2 (before injection on D+2), and D+3 ( Figure 4 A).

[0291] After paw incision, vehicle-treated mice developed mechanical allodynia (see Figure 4 In contrast, mice treated with "peptide 1+2" did not develop mechanical allodynia. Compared with vehicle-treated mice, mice treated with "peptide 1+2" showed statistically significantly higher response thresholds at D+1, D+2, and D3 ( Figure 4 B).

[0292] These results indicate that the group of peptides of the present invention can be used as analgesics in the paw incision model (a postoperative pain model).

[0293] Example 3: Lack of tolerance in the chronic constriction injury (CCI) neuropathic pain model.

[0294] The purpose of this experiment was to evaluate whether subjects treated with a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("Peptide 1+2") could induce tolerance to the group of peptides following repeated subcutaneous administration of the group of peptides.

[0295] The CCI model was placed at the site according to the above-mentioned protocol. Starting from D7 after surgery, mice were subcutaneously injected with "Peptide 1+2" (0.3 mg / kg) twice a day for 14 consecutive days. Mechanical sensitivity was tested by the up / down method using Von Frey filaments before and 7 days after surgery to verify CCI-induced mechanical allodynia. The time course response was performed after the first application. Then, the analgesic effect of "Peptide 1+2" was tested every other day. Then, after the last application (D20), a complete time course response ( Figure 5 A). The last measurement was performed at D+30 (10 days after the end of treatment) to verify the proper recovery of the mice.

[0296] The first subcutaneous injection of "Peptide 1+2" induced a strong increase in the response (i.e., paw withdrawal) threshold, demonstrating the analgesic effect of "Peptide 1+2" ( Figure 5 B- Comparison between the response measured in the “prodrug” condition and the response measured two hours after the first injection administration). Interestingly, the analgesic effect of “peptide 1+2” remained constant throughout the duration of treatment (14 days), without any decrease in efficacy, even at the last injection corresponding to D20 ( Figure 5 B, There is no significant difference between the response measured as the first injection compared to the response measured at the last injection). These results indicate that the peptides of the invention can be used repeatedly over a long period of time without inducing tolerance in the subject.

[0297] Example 4: Comparison of different doses in the treatment of postoperative pain model - preventive effect

[0298] The purpose of this experiment was to test different doses of a set of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 ("Peptide 1+2") for treating subjects with postoperative pain when administered subcutaneously in vivo in a postoperative pain model.

[0299] "Peptide 1+2" or vehicle was administered subcutaneously twice daily according to three different schedules. "D-1 / D0" schedule: administration was performed one day before surgery (D-1) and on the day of surgery (D0); "D0" schedule: administration was performed on the day of surgery (D0); "D0 / D+1 / D+2" schedule: administration was performed one day before surgery (D-1), on the day of surgery (D0), and on D+1 and D+2 days after surgery. The administration of D0 was performed 1 hour before surgery and 1 hour after surgery (and waking up), respectively. Mechanical threshold response measurements were performed on D+1 and D+2 before the administration of the two peptide groups or vehicle ( Figure 6 A).

[0300] After paw incision, vehicle-treated mice developed mechanical allodynia (see Figure 6 D+1, D+2 and D+3 in B). In contrast, mice treated with "peptide 1+2" did not develop mechanical allodynia, regardless of the regimen used. Interestingly, mice treated with the "D-1 / D0" and "D0" regimens, which were not treated after surgery, did not develop mechanical allodynia, indicating that the group of peptides of the present invention can be used prophylactically in the paw incision model (postoperative pain model).

[0301] Example 5: Analgesic effect in an inflammatory pain model

[0302] The purpose of this experiment was to evaluate the analgesic effect of a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (peptide 1+2) in vivo in an inflammatory pain model (capsaicin inflammatory pain model) by intravenous administration.

[0303] The experiments were performed on eight-week-old male WT C57Bl6 mice. Two groups of 8 mice were used. Peptide 1+2 was resuspended in HMPC solution at a concentration of 30 μg / mL and 10 μl was injected per gram.

[0304] Capsaicin (Sigma) stock solution (dissolved in 16.7 mg / mL anhydrous ethanol) was diluted in 0.9% NaCl at 0.25 mg / mL, and then 10 μL was injected into the plantar surface of the left hind paw of the mouse using a Hamilton syringe and a 26G needle. As previously described, the baseline was determined by measuring with a Von Frey filament by the up / down method. 24h after the injection of capsaicin (D+1), two groups of mice were treated with "peptide 1+2" or vehicle, respectively. The reaction threshold was measured 1 hour (+1h), 2 hours (+2h) and then 4 hours (+4h) after subcutaneous administration of 0.3 mg / kg of "peptide 1+2" solution (n=8) or vehicle solution (n=8).

[0305] 24 h after capsaicin injection, mice developed mechanical allodynia (see D+1- Figure 7 ), while the group to which the peptide of the present invention was subcutaneously administered induced a statistically significant increase in the response threshold 2 hours after administration.

[0306] These results indicate that the group of peptides of the present invention leads to analgesic effects via subcutaneous administration in the capsaicin model (inflammatory pain model).

[0307] Example 6: Analgesic effect in osteoarthritis pain model

[0308] The aim of this experiment was to evaluate the analgesic effect of a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (peptide 1+2) administered subcutaneously in vivo in an osteoarthritis pain model (monoiodoacetate).

[0309] Two groups of mice were used, 8 in each group. Mechanical sensitivity was tested by the up / down method using Von Frey filaments 14 days before and after monoiodoacetate (MIA) was injected into each group. 24h after MIA, two groups of mice were treated with "peptide 1+2" or vehicle (NaCl solution). Time course reactions were performed to track the analgesic effect of "peptide 1+2". The reaction threshold was measured 1 hour (+1h), 2 hours (+2h) and then 4 hours (+4h) after applying "peptide 1+2" or vehicle.

[0310] At day 14 after MIA injection, all mice showed a dramatic decrease in mechanical threshold compared with baseline thresholds, indicating a strong mechanical hypersensitivity ( Figure 8 Subcutaneous administration of the vehicle had no analgesic effect. In contrast, the analgesic effect of "peptide 1+2" was statistically significant compared with the negative control (vehicle) at 1 and 2 hours after administration ( Figure 8 ).

[0311] These results indicate that the group of peptides of the present invention results in an analgesic effect via subcutaneous administration in an arthritis model.

[0312] Example 7: Analgesic effect in frostbite pain model.

[0313] The purpose of this experiment was to evaluate the analgesic effect of a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (peptide 1+2) administered subcutaneously in vivo in a frostbite pain model.

[0314] Two groups of mice were used, 16 in each group. Each group had 8 males and 8 females. Mechanical sensitivity was tested by the up / down method using Von Frey filaments before (baseline) and 24 h after cold exposure (D+1). After administration of "peptide 1+2" or vehicle (NaCl solution) 24 h after cold exposure, a time course reaction was performed to track the analgesic effect of "peptide 1+2" 1 hour (D1+1), 2 hours (D1+2h) and 4 hours (D1+4h) after administration.

[0315] At 24 h after cold exposure, all mice showed a dramatic decrease in mechanical threshold compared to baseline thresholds, indicating a robust mechanical hypersensitivity ( Fig. 9 Subcutaneous administration of the vehicle had no analgesic effect. In contrast, the analgesic effect of "peptide 1+2" was statistically significant compared with the negative control (vehicle) at 1 and 2 hours after administration ( Fig. 9 ).

[0316] These results indicate that the group of peptides of the present invention leads to an analgesic effect via subcutaneous administration in the frostbite (or ice burn) model. This model is also considered a type of acute pain model.

[0317] Example 8: Analgesic effect in ultraviolet burn pain model

[0318] The purpose of this experiment was to evaluate the analgesic effect of a group of peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (peptide 1+2) administered subcutaneously in vivo in the UV burn pain model (also known as the sunburn pain model).

[0319] Two groups of mice were used, 16 in each group. Each group had 8 males and 8 females. Mechanical sensitivity was tested by the up / down method using Von Frey filaments before (baseline) and 24h after UV irradiation (D+1). After administration of "peptide 1+2" or vehicle (NaCl solution), a time course reaction was performed to track the analgesic effect 1 hour (D1+1h), 2 hours (D1+2h) and 4 hours (D1+4h) after administration of "peptide 1+2".

[0320] At 24 h after UV exposure, all mice showed a dramatic decrease in mechanical threshold compared to baseline thresholds, indicating a strong mechanical hypersensitivity ( Fig.10Subcutaneous administration of the vehicle had no analgesic effect. In contrast, the analgesic effect of the group "peptide 1+2" was statistically significant compared with the negative control (vehicle) at 1 and 2 hours after administration ( Fig.10 ).

[0321] These results indicate that the group of peptides of the present invention leads to an analgesic effect via subcutaneous administration in the UV burn (or sun burn) pain model. This model is also considered a type of acute pain model.

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Claims

1. A composition comprising a first peptide and a second peptide, wherein the first peptide is SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1, and the second peptide is SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2, wherein the two peptides are not connected to each other.

2. The composition of claim 1, wherein if the composition comprises a peptide having at least 90% identity to SEQ ID NO:1 and / or a peptide having at least 90% identity to SEQ ID NO:2, the glutamine (Q) residue at position 13 in the peptide of SEQ ID NO:1 remains unchanged in the amino acid sequence of the peptide having at least 90% identity to SEQ ID NO:1, and the tyrosine (Y) residue at position 18 in the peptide of SEQ ID NO:2 remains unchanged in the amino acid sequence of the peptide having at least 90% identity to SEQ ID NO:2 or is replaced by serine (S).

3. A group of components or compositions comprising nucleic acids allowing the expression of two peptides that are not linked to each other, wherein the first nucleic acid comprises a sequence encoding the peptide of SEQ ID NO: 1 or a peptide that is at least 90% identical to SEQ ID NO: 1, and the second nucleic acid comprises a sequence encoding the peptide of SEQ ID NO: 2 or a peptide that is at least 90% identical to SEQ ID NO:

2.

4. A set of components or compositions, comprising vectors allowing the expression of two peptides that are not linked to each other, wherein the first vector comprises a sequence encoding the peptide of SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1, and the second vector comprises a sequence encoding the peptide of SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO:

2.

5. A cell or a composition comprising the cell and an acceptable support, wherein the cell allows the expression of two peptides that are not connected to each other, and wherein the cell comprises a first nucleic acid sequence or a vector encoding a peptide of SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1, and a second nucleic acid sequence or a vector encoding a peptide of SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO:

2.

6. The composition of any one of claims 1 to 5, wherein the composition further comprises at least one additional active compound, preferably an active compound effective against pain, even more preferably a SAID, NSAID or an opioid.

7. The composition of any one of claims 1-6, wherein the composition comprises a pharmaceutically and / or dietary acceptable support.

8. The composition of any one of claims 1-7, wherein the composition further comprises ascorbic acid or an ascorbate salt.

9. A group of a first peptide and a second peptide, a group as described in claim 3 or 4, a composition as described in any one of claims 1-8, or a cell as described in claim 5, wherein the first peptide is SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1, and the second peptide is SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2, wherein the two peptides are not connected to each other, wherein the group, composition or cell is used as a drug for a subject in need thereof.

10. A group of a first peptide and a second peptide, a group as described in claim 3 or 4, a composition as described in any one of claims 1-8, or a cell as described in claim 5, wherein the first peptide is SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1, and the second peptide is a second peptide of SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2, wherein the two peptides are not connected to each other, wherein the group, composition or cell is used to prevent or treat pain in a subject in need thereof.

11. The group, composition or cell for use of claim 10, wherein the pain is chronic pain, neuropathic pain, postoperative pain, inflammatory pain, arthritic pain, pain associated with joint injury, pain associated with Ehlers-Danlos syndrome, pain caused by sunburn, pain caused by ice burns, hyperalgesia, allodynia or acute pain.

12. The group, composition or cell for use according to any one of claims 9 to 11, wherein the subject is a mammal, especially a human.

13. The group or composition for use according to any one of claims 9 to 12, wherein the composition is administered to the subject intramuscularly, intravenously, intraperitoneally, orally (perorally), peranally, dermally, subcutaneously, topically, transdermally, transdermally or intrathecally, preferably subcutaneously or orally.

14. The set or composition for use according to any one of claims 9 to 13, wherein the set or composition comprises 1 μg to 100 mg, preferably 25 μg to 10 mg, of the first peptide and the second peptide per unit dose.

15. The group or composition for use according to any one of claims 9 to 13, wherein the group or composition is administered to a mammal at a dose of 1 μg / kg / day to 100 mg / kg / day, preferably 2.5 μg / kg / day to 0.6 mg / kg / day.

16. A kit comprising i) a first peptide, wherein the first peptide is SEQ ID NO: 1 or a peptide having at least 90% identity with SEQ ID NO: 1, or a nucleic acid sequence encoding the peptide, ii) a second peptide, wherein the second peptide is SEQ ID NO: 2 or a peptide having at least 90% identity with SEQ ID NO: 2, or a nucleic acid encoding the peptide, the first peptide and the second peptide or the nucleic acid encoding the peptide are in different containers, and optionally iii) written instructions for using the kit.

Images