Peptides and pharmaceutical composition as an analgesic

Abstract

The present invention is within the field of biomedicine. Specifically, the present invention relates to the use of peptides comprising the amino acid sequence SEQ ID NO:1, or a pharmaceutical composition thereof, as an analgesic, in other words, in treating pain in a subject.

Description

[0001] DESCRIPTION

[0002] Peptides and pharmaceutical composition as an analgesic

[0003] The present invention falls within the field of biomedicine. Specifically, the present invention relates to the use of peptides comprising the amino acid sequence SEQ ID NO: 1, or a pharmaceutical composition thereof, as an analgesic, that is, in the treatment of pain in a subject.

[0004] BACKGROUND OF THE INVENTION

[0005] Pain is the most common reason for medical consultations and the most common reason for requesting over-the-counter medications. Various surveys conducted in our country show that around 30% of the population reports having experienced it in the last 48 hours, more than 40% in the last week, and almost 80% in the last six months. Both its prevalence and intensity are higher in women and also increase with age.

[0006] Pain classification can be done based on various criteria:

[0007] According to its duration: Acute pain (a short-term phenomenon that is generally associated with tissue damage and disappears with the healing of the latter), chronic pain (lasting more than 3 or 6 months, extending beyond the healing of the injury that caused it or associated with a chronic condition).

[0008] According to its origin: Nociceptive (caused by the activation of pain receptors (nociceptors) in response to a stimulus (injury, inflammation, infection, disease); Neuropathic (originated by a direct stimulus of the central nervous system (CNS) or an injury to the peripheral nerves. It is common for it to be disproportionate to the stimulus that produces it (hyperalgesia) and to appear without an identifiable cause. It is considered pathological pain, since it is of no use as a warning or defense mechanism); Psychogenic (not due to nociceptive stimulation or a neuronal alteration, but has a psychic cause (depression, hypochondria) or it is the disproportionate intensification of an organic pain that is due to psychological factors).

[0009] According to its location: a) Nociceptive pain is divided into: • Somatic pain, when receptors in the skin, musculoskeletal system, or vascular system are stimulated. It is characterized by being well localized and, although it is often sharp, its typology varies from one patient to another.

[0010] • Visceral pain, which is due to injuries or dysfunctions of internal organs, although some organs do not experience pain, such as the liver or lungs. It is deep, continuous, and poorly localized, radiating even to areas far from the source. It is often accompanied by autonomic symptoms (nausea, vomiting, sweating). b) Neuropathic pain is divided into central and peripheral, depending on which part of the nervous system the injury or disease causing it is located in.

[0011] Neuropathic pain is characterized by a neurological injury or dysfunction (central or peripheral nervous system) that involves a disorder of the nerve fibers. However, it is, in any case, a sensory phenomenon involving abnormal activity in the sensory transmission pathways. Neuropathic pain is defined by the IASP (International Association for the Study of Pain) as "pain that originates as a direct consequence of an injury or disease affecting the somatosensory system" (Jensen and Gebhart, 2008, Pain, 140:399-400).

[0012] Neuropathic pain is associated with multiple diseases with very different causes, including diabetes. Peripheral neuropathy is the most frequent symptomatic complication in patients with diabetes, where chronic hyperglycemia plays a fundamental role in its pathophysiology by promoting microvascular lesions and a proinflammatory state in different parts of the system. The management of diabetic neuropathic pain is complex, as symptoms are not always controlled due to its multifactorial etiology. The use of chronic nonsteroidal anti-inflammatory drugs (NSAIDs) is limited by the risk of nephrotoxicity, especially in diabetes (Samper Bernal et al., 2010, Rev Soc Esp Dolor, 17:286-296).Drugs such as catecholamine and serotonin reuptake inhibitors (antidepressants), antiepileptics and opioids offer pain relief, although possible adverse effects must be monitored since there is no single exclusive analgesic treatment, which accentuates the severity of this treatment (Samper Bernal et al., 2010, Rev Soc Esp Dolor, 17:286-296; Bril et al., 2011, Neurology, 76:1758-1765; Botas Velasco, 2017, Angiología, 69:174-181).

[0013] Neuropathic pain has been shown to be at least partly dependent on N-methyl-D-aspartate (NMDA) receptors (Deng et al., 2019, Cell Mol Life Sci, 76:1889–1899). However, the clinical use of NMDA receptor blockers is limited by side effects resulting from the suppression of their physiological functions.

[0014] Therefore, there is a need in the state of the art for new therapeutic strategies to suppress pain hypersensitivity without blocking NMDA receptors, thus avoiding undesirable side effects.

[0015] DESCRIPTION OF THE INVENTION

[0016] The inventors have identified that short amino acid sequences of connexin 43 (Cx43) capable of inhibiting the activity of Src (non-receptor protein tyrosine kinase encoded by the first described oncogene Src), have the property of lessening, diminishing or reducing the pain suffered by a subject, being capable of acting as an analgesic. In particular, the inventors have observed that the sequence 266-283 of Cx43 (See Example 1 of the present description) is capable of lessening, diminishing, reducing or eliminating neuropathic pain inflicted in rats. To this end, the inventors subcutaneously administered the peptide to be tested in rats with diabetic neuropathy and, using the heat focus technique, they verified that these rats took longer to react to pain, compared to rats that had not received the peptide in question (see Figure 3). Therefore, the present invention provides compounds useful in the prevention and / or treatment of pain.

[0017] Thus, in one aspect, the present invention relates to a peptide comprising, or consisting of, the sequence SEQ ID NO: 1 (hereinafter “peptide of the invention”) for use in the prevention and / or treatment of pain, that is, it can act as an analgesic (hereinafter “use of the peptide of the invention”).

[0018] The sequence SEQ ID NO: 1 (AYFNGCSSPT) corresponds to the sequence between amino acids 266-275 of Cx43, where said amino acids 266-275 are within the carboxyl terminal end of Cx43 (represented by the sequence SEQ ID NO: 2) and in which the Src inhibitory activity of said terminal end resides.

[0019] The sequence SEQ ID NO: 2 corresponds to the carboxyl terminus of Cx43 and comprises the following amino acid sequence: FFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLV TGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKK LAAGHELQPLAIVDQRPSSRASSRASSRPRPDDLEI Thus, without wishing to be bound by any theory, the inventors think that if the sequence 266-283 of Cx43 (SEQ ID NO: 3: AYFNGCSSPTAPLSPMSP) has analgesic properties (see Example 1), it is plausible that smaller sequences containing the critical residues for the Src inhibitory activity of the carboxyl terminus of Cx43, such as amino acids 266-275 of Cx43 [SEQ ID NO: 1 - AYFNGCSSPT (see WO2023139156)], also possess such analgesic capacity.

[0020] Therefore, any peptide that comprises in its amino acid sequence SEQ ID NO: 1 (0x43266-275) has the ability to function as an analgesic and is susceptible to being used in the prevention and / or treatment of pain.

[0021] Thus, in another particular embodiment of the use of the invention, the peptide of the invention comprises, or consists of, the sequence SEQ ID NO: 3 (AYFNGCSSPTAPLSPMSP).

[0022] In a particular embodiment of the use of the invention, the peptide of the invention comprises, or consists of, the amino acid sequence SEQ ID NO: 4 (0x43266-277) [AYFNGCSSPTAP1.

[0023] In another particular embodiment of the use of the invention, the peptide of the invention comprises, or consists of, the amino acid sequence SEQ ID NO: 5 (0x43266-279) [AYFNGCSSPTAPLS1.

[0024] In another particular embodiment of the use of the invention, the peptide of the invention comprises, or consists of, the amino acid sequence SEQ ID NO: 6 (Cx43266-28i) [AYFNGCSSPTAPLSPM1.

[0025] In the context of the present invention, a peptide is understood to be a molecule formed by joining between 6 and 150 amino acids by means of peptide bonds. However, in a particular embodiment, the peptide of the invention has a length of between 10 and 100 amino acids (including the ends), more particularly between 10 and 50 amino acids (including the ends), between 10 and 40 amino acids (including the ends), between 10 and 30 amino acids (including the ends) or between 10 and 20 amino acids (including the ends). In an even more particular embodiment, the peptide of the invention has a length of between 10 and 16 amino acids (including the external ones). As understood by those skilled in the art, the smaller the peptide, the easier it will be to administer and the fewer side effects it will present.

[0026] The peptide of the invention can be obtained by techniques widely known in the art. Examples of peptide production techniques include, but are not limited to, chemical or biological synthesis, genetic recombination, or expression of polynucleotides encoding the peptide of the invention.

[0027] Additionally, the carboxyl and amino termini of the peptide of the invention may be protected against proteolysis. For example, the amino terminus may be in the form of an acetyl group and / or the carboxyl terminus may be in the form of an amide group. It is also possible to carry out internal modifications of the peptides to make them resistant to proteolysis, for example, in which at least one peptide bridge -CONH- is modified and replaced by a reduced bond (CH2NH), a retroinverso bond (NHCO), an oxymethylene bond (CH2-O), a thiomethylene bond (CH2-S), a ketomethylene bond (CO-CH2), a hydroxyethylene bond (CHOH-CH2), a bond (NN), an E-alcene bond or a -CH=CH- bond. The amino acids of the peptide of the invention may be in the D-configuration, which can give rise to peptides resistant to proteolysis.Peptides may also be stabilized by intramolecular crosslinking, for example, by modifying at least two amino acid residues with olefin side chains, preferably C3-C8 alkenyl chains, preferably pentel-2- ¡I chains, followed by crosslinking of the chains as described in the so-called “staple” technology (Walensky et al., 2004, Science 205: 1466-1470). All such chemically modified peptides to resist proteolysis are also contemplated within the present invention.

[0028] Additional modifications to the peptide of the invention include covalent attachment to a polyethylene glycol (PEG) molecule at its carboxyl terminus or to a lysine residue, in order to reduce its urinary elimination and the therapeutic dose, and to increase the half-life of the peptide in blood plasma. The half-life of the peptide can also be increased by including the peptide in a biodegradable and biocompatible polymeric material to form microspheres that are used as a drug delivery system. Polymers and copolymers are, for example, poly (D,L-lactide-co-glycolic acid) or PLGA. The techniques and methods for manufacturing lipid microspheres or nanocapsules for use in drug delivery are widely known to those skilled in the art. Any other method of targeted drug delivery can be used in the context of the present invention.Likewise, the peptide of the invention may comprise conservative amino acid substitutions in its amino acid sequence that maintain the peptide's ability to act as an analgesic. Therefore, in the context of the present invention, peptides derived from the peptide of the invention, known as "vahantes," are also contemplated. Although they do not have 100% sequence identity with the peptide of the invention, they retain their ability to act as an analgesic, since the amino acids have been replaced by other biologically similar ones. Assays to determine whether a peptide derived from the peptide of the invention has the ability to act as an analgesic are described in Example 1 of the present description.

[0029] Thus, in a particular embodiment, the peptide of the invention comprises, or consists of, an amino acid sequence with a sequence identity of at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with SEQ ID NO: 1.

[0030] In another particular embodiment, the peptide of the invention comprises, or consists of, an amino acid sequence with a sequence identity of at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 26.

[0031] The peptide of the invention may be linked to a cellular internalization sequence. The peptide may be introduced into the cell by any of the methods known in the art, including, but not limited to, direct injection, electroporation, or transfection. However, these techniques are relatively complex and have limitations when applied in vivo and accessing the entire tumor cell population. If the peptide is to be administered to a subject, the peptide may be introduced into the cell using gene therapy techniques through the use of viral vectors, or through cellular internalization sequences that allow the peptide to cross the plasma membrane.

[0032] Thus, in a particular embodiment, the peptide of the invention is covalently linked to a sequence of amino acids for cellular internalization.

[0033] In the present invention, "cellular internalization amino acid sequences" or "cellular internalization sequences" or "cell-penetrating peptides" (CPPs) are understood to be amino acid sequences that possess the ability to transport molecules across the plasma membrane without losing their integrity. Among the most commonly used sequences are TAT, Antennapedia (Antp), and oligo-arginines, whose common characteristic is the presence of cationic amino acid groups. These internalization sequences allow the peptide to be internalized directly into the cell.

[0034] <h2 style=";text-align:left;direction:ltr">Ejemplos de secuencias de internalización celulares, pero no se limitan a, RQIKIWFQNRRMKWKK (SEQ ID NO: 12), VKKKKIKREIKI (SEQ ID NO: 13), FFLIPKG (SEQ ID NO: 14), SMoCs [Okuyama et al., 2007. Nature Methods, 4, 153 - 159], YGRKKRRQRRR (SEQ ID NO: 7), DSLKSYWYLQKFSWR (SEQ ID NO: 15), KLWMRWWSPTTRRYG (SEQ ID NO: 16), RLWMRWYSPWTRRWG (SEQ ID NO: 17), RLIMRIYAPTTRRYG (SEQ ID NO: 18), RLYMRYYSPTTRRYG (SEQ ID NO: 19), RLWMRWYSPRTRAYG (SEQ ID NO: 20), KRPTMRFRYTWNPMK (SEQ ID NO: 21), WKCRRQCFRVLHHWN (SEQ ID NO: 22), WKCRRQAFRVLHHWN (SEQ ID NO: 23), WKARRQAFRVLHHWN (SEQ ID NO: 24), the penetrant sequence in glioma cells (Berges et al. Píos One 2012; 7(11):e49436) and Biotin-YSSYSAPVSSSLSVRRSYSSSSGS-CONH2 (SEQ ID NO: 25).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0035] In a particular embodiment of the peptide of the invention for the use of the invention, the cellular internalization sequence comprises, or consists of, the amino acid sequence SEQ ID NO: 7 (YGRKKRRQRRR). Throughout this document, the term “TAT sequence” or “TAT” is also used to refer to SEQ ID NO: 7.

[0036] The cellular internalization sequence can be linked to the peptide of the invention at either the amino terminus or the carboxyl terminus of the peptide of the invention. However, in a particular embodiment, the cellular internalization sequence is linked to the amino terminus of the peptide.

[0037] Thus, in the present invention, the peptides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, can be found linked to a cellular internalization sequence.

[0038] In a particular embodiment, the peptide of the invention comprises, or consists of, the amino acid sequence SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. SEQ ID NO : 8 - YGRKKRRQRRRAYFNGCSSPT

[0039] SEQ ID NO : 9 - YGRKKRRQRRRAYFNGCSSPTAP

[0040] SEQ ID NO : 10 - YGRKKRRQRRRAYFNGCSSPTAPLS

[0041] SEQ ID NO : 11 - YGRKKRRQRRRAYFNGC SS PT APL S PM

[0042] SEQ ID NO : 26 YGRKKRRQRRRAYFNGCSSPTAPLSPMSP

[0043] As previously explained, in the present invention we refer with “peptide of the invention” to a peptide comprising the amino acid sequence SEQ ID NO: 1. Thus, a peptide comprising the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 26, as well as their functionally equivalent variants, is encompassed under the term “peptide of the invention” or “peptides of the invention”, terms used interchangeably in this document.

[0044] As previously indicated, the peptide of the invention can be obtained by techniques widely known in the art, such as the expression in a cell of the polynucleotide encoding the peptide of the invention, and its subsequent isolation. Thus, in another aspect, the invention relates to a polynucleotide, hereinafter the "polynucleotide of the invention," that encodes the peptide of the invention, for use in the prevention and / or treatment of pain.

[0045] The term "polynucleotide," as used herein, refers to a polymeric form of nucleotides of any length, composed of ribonucleotides and / or deoxyribonucleotides. The term includes both single-stranded and double-stranded polynucleotides, as well as modified (methylated, protected, and the like) polynucleotides. The polynucleotide of the invention may be DNA, RNA, or cDNA.

[0046] In another aspect, the invention relates to a gene construct, hereinafter the “gene construct of the invention”, comprising the polynucleotide of the invention, for use in the prevention and / or treatment of pain.

[0047] Preferably, the construct comprises the polynucleotide of the invention operably linked to sequences regulating the expression of the polynucleotide of the invention. In principle, any promoter can be used in the gene constructs of the present invention, provided that said promoter is compatible with the cells in which the polynucleotide is to be expressed.

[0048] A promoter, or promoter region, is a nucleotide sequence that controls the transcription of a given gene (nucleotide sequences). In the present invention, it refers to a nucleotide sequence that controls the transcription of the polynucleotide of the invention. Promoter sequences can be unidirectional or bidirectional. A unidirectional promoter is one that controls the transcription of one or more genes that are located in tandem with the first. "In tandem" refers to the 3' end of the first gene being followed, either consecutively or separated by a specific nucleotide sequence, by the 5' end of the second gene. A bidirectional promoter refers to the promoter region that controls transcription in two opposite directions, for example the sequence preceding the kivD gene of L. lactis IFPL730 which acts as a bidirectional promoter.That is, a bidirectional promoter directs the transcription of two genes located divergently, that is, in opposite directions, with the 5' end of both nucleotide sequences being closer to each other than the 3' end. In the present invention, the terms "promoter" and "promoter region" are used interchangeably. Furthermore, the promoters in the present invention can be constitutive or inducible. The term "inducible", as used in the present description, refers to the possibility of the promoter having a control element that allows the transcription of the gene it regulates to be activated or deactivated (repressed), in the presence of a factor external to the promoter.

[0049] Suitable promoters for carrying out the present invention include, but are not limited to, constitutive promoters such as those derived from the genomes of eukaryotic viruses such as polyoma virus, adenovirus, SV40, CMV, avian sarcoma virus, hepatitis B virus, the metallothionein gene promoter, the herpes simplex virus thymidine kinase gene promoter, LTR regions of retroviruses, the immunoglobulin gene promoter, the actin gene promoter, the EF-1 alpha gene promoter as well as inducible promoters in which protein expression is dependent on the addition of an exogenous molecule or signal, such as the tetracycline system, the NFKB / light W system, the Cre / Lox system and the heat shock gene promoter, regulatable RNA polymerase II promoters as well as tissue specific promoters.

[0050] Additionally, the gene construct of the invention may contain markers or tags that allow the isolation of the peptide of the invention once it is synthesized in the cell.

[0051] Furthermore, the polynucleotide or gene construct of the invention may be part of a vector. Thus, in another aspect, the invention relates to a vector, hereinafter "vector of the invention," comprising the polynucleotide or gene construct of the invention, for use in the prevention and / or treatment of pain.

[0052] As understood by one skilled in the art, the type of vector used may be a cloning vector suitable for propagation or an expression vector. Thus, examples of suitable vectors according to the present invention include, but are not limited to, higher eukaryotic cell expression vectors based either on viral vectors (adenoviruses, adeno-associated viruses as well as retroviruses and lentiviruses) as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1 / hyg pHCMV / Zeo, pCR3.1, pEFVHis, pIND / GS, pRc / HCMV2, pSV40 / Ze02, pTRACER HCMV, pUB6N5-His, pVAXI, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTI.

[0053] In a particular embodiment, the vector of the invention for use in the prevention and / or treatment of pain is a viral vector, preferably a viral vector of a retrovirus, a lentivirus or an adenovirus.

[0054] The vector of the invention can be used to transform, transfect or infect cells susceptible to being transformed, transfected or infected by said vector.

[0055] The vector into which the nucleotide sequence, preferably DNA, is introduced can be a plasmid or a vector that, when introduced into a host cell, integrates into the genome of said cell and replicates along with the chromosome(s) into which it has integrated. Such a vector can be obtained by conventional methods known to those skilled in the art.

[0056] Therefore, another aspect of the invention relates to a nanoparticle, hereinafter the “nanoparticle of the invention”, comprising the polynucleotide, the gene construct or the vector of the invention, for use in the prevention and / or treatment of pain.

[0057] The term “nanoparticle,” as used herein, refers to any material having dimensions in the range of 1-1,000 nm. In some embodiments, nanoparticles have dimensions in the range of 2-200 nm, preferably in the range of 2-150 nm, and even more preferably in the range of 2-100 nm. Nanoparticles can contribute to preserving the integrity of the peptide, polynucleotide, vector, or cell of the invention until it reaches the target organ. Likewise, nanoparticles can also be modified to include moieties that allow the nanoparticle to be directed to an organ of interest. The nanoparticles that can be used in the context of the present invention can be formed from any biomaterial that allows the structure and functionality of the active ingredient (peptide, polynucleotide, vector, or cell of the invention) to be maintained and released in the target organ.The most commonly used biomaterials in nanoparticle manufacturing are polymer composites such as alginate, chitosan, gelatin, hyaluronic acid, poly(lactic-co-glycolic acid) (PLGA), polylactide (PLA), polycaprolactone (PCL), and polyanionic cellulose (PAC). Their bioavailability, improved encapsulation, release control, and cyto- and biocompatible properties enhance the therapeutic value of the encapsulated agents.

[0058] In a particular embodiment of the nanoparticle of the invention for use in the prevention and / or treatment of pain in a subject, the nanoparticle is a nanosphere (spherical shaped nanoparticle), a nanocapsule, a micelle, a liposome or a dendrimer.

[0059] In the present invention, the term “nanospheres” refers to a spherical matrix system, which has the active agent dispersed in a polymeric matrix in a homogeneous manner.

[0060] In the present invention, the term “nanocapsules” refers to the vesicular system that surrounds the active agent within a cavity surrounded by a polymeric shell, which controls its release depending on its nature.

[0061] In the present invention, the term "micelle" refers to the conglomerate of molecules that constitutes one of the phases of colloids, and within which the active ingredient is encapsulated.

[0062] In the present invention, the term "liposome" refers to a spherical vesicle (small bubble) with a membrane composed of a double layer of phospholipids, consisting of water-soluble and fat-soluble parts. In the present invention, the term "dendrimer" refers to a three-dimensional molecule of tree-like construction and polymeric nature, where the distribution of the molecules that constitute the linear polymers has a chemical structure that presents a form of generational growth, G0, G1, G2.

[0063] The materials and methods for the manufacture of nanoparticles and the encapsulation of active ingredients within them are widely known in the state of the art and are routine practice for those skilled in the art.

[0064] The inventors have identified that the peptide of the invention is capable of acting as an analgesic, being useful in the prevention and / or treatment of pain in a subject. Furthermore, the peptide, polynucleotide, gene construct, vector, or nanoparticle of the invention can be part of a composition, preferably a pharmaceutical composition.

[0065] Thus, another aspect of the invention relates to a composition, hereinafter the "composition of the invention," comprising the peptide, polynucleotide, gene construct, vector, or nanoparticle of the invention, for use in the prevention and / or treatment of pain in a subject. In a particular embodiment, the composition of the invention is a pharmaceutical composition.

[0066] In a particular embodiment, the composition of the invention comprises the peptide, polynucleotide, gene construct, vector or nanoparticle of the invention in a therapeutically effective amount, and a vehicle which, in the case of a pharmaceutical composition, will be a pharmaceutically acceptable vehicle.

[0067] In the present invention, "pharmaceutical composition" refers to any preparation or pharmaceutical form whose composition formula, expressed in units of the international system, consists of a substance or mixture of substances, with constant weight, volume, and percentages, prepared in legally established pharmaceutical laboratories, packaged, or labeled to be distributed and marketed as effective for the diagnosis, treatment, mitigation, and prophylaxis of a disease, physical anomaly, or symptom, or the restoration, correction, or modification of the balance of organic functions in humans and animals. The preparation of the pharmaceutical composition may be carried out by any of the methods described in the state of the art.As used in this description, the term "therapeutically effective amount" refers to the quantity of the compound or pharmaceutical composition of the invention that produces the desired effect and, in general, will be determined, among other things, by the characteristics of said compound or pharmaceutical composition and the therapeutic effect to be achieved. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, or tolerance of the subject. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are those known in the state of the art.

[0068] The term "vehicle" refers to a diluent or excipient with which the active ingredient is administered. Such vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as vehicles, particularly for injectable solutions. Preferably, pharmaceutical vehicles are approved by a state or federal regulatory agency or are listed in the United States Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, and more particularly in humans.The vehicles and auxiliary substances required to manufacture the desired pharmaceutical form of administration of the pharmaceutical composition of the invention will depend, among other factors, on the pharmaceutical form of administration chosen. These pharmaceutical forms of administration of the pharmaceutical composition will be manufactured according to conventional methods known to those skilled in the art.

[0069] Furthermore, in the present invention, it is contemplated that the peptides of the invention may be associated with delivery systems or molecular vehicles, including, but not limited to, exosomes and microvesicles. These delivery systems or molecular vehicles are capable, thanks to their properties, of crossing cell membranes and delivering their "cargo" (in the present invention, the peptides of the invention), in a biologically active form, and can also cross biological membranes such as the blood-brain barrier. Thus, in a preferred embodiment, the composition of the invention also comprises exosomes and / or microvesicles. The compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including a human, in a variety of forms known in the state of the art. Thus, they can be, but are not limited to, in aqueous or non-aqueous solutions, emulsions, or suspensions.Examples of non-aqueous solutions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable organic esters such as ethyl oleate. Examples of aqueous solutions include, but are not limited to, water, alcoholic solutions in water, or saline media. Aqueous solutions may be buffered or unbuffered and may have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelating agents, or the like, or nutrients including glucose, dextrose, vitamins, and minerals. Alternatively, the compositions may be prepared for administration in solid form.The compositions may be combined with various inert carriers or excipients, including but not limited to: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose; dispersing agents, such as alginic acid or corn starch; lubricants, such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or flavoring agents, such as peppermint or methyl salicylate.

[0070] Additionally, the composition of the invention may comprise an adjuvant. By "adjuvant" is meant any substance that enhances the effectiveness of the pharmaceutical composition of the invention. Examples of adjuvants include, but are not limited to, adjuvants formed from aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate; oil-in-water or water-in-oil emulsion formulations such as Complete Freund's Adjuvant (CFA) as well as Incomplete Freund's Adjuvant (IFA); mineral gels; block copolymers, Avhdine™, SEAM62; adjuvants formed from bacterial cell wall components such as adjuvants including liposaccharides (e.g.,lipid A or Lipid A monophosphohl (MLA), trehalose dimycolate (TDM), and cell wall skeleton components (CWS), heat shock proteins or their derivatives, adjuvants derived from bosylated bacterial toxins ADPh, including diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), the heat-labile E. coli toxins (LT1 and LT2), Pseudomonas endotoxin A and exotoxin, B. cereus exoenzyme B, B. sphaerieus toxin, C. botulinum toxins C2 and C3, C. limosum exoenzyme as well as the toxins of C. perfringens, C. spiriforma and C. diffieile, S.aureus, EDIM and mutant toxin mutants such as CRM-197, a mutating toxin not toxic to diphtheria; saponinas como las ISCOMs (complejos inmunoestimuladores), kemocinas quimioquinas y citoquinas como la interleuquinas (IL-I IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 12, etc), interferones (como el interferon gama) factor estimulador de colonias de macrófagos (M-CSF), Factor de necrosis tumoral (TNF), defensinas 102, RANTES, MIPI - .alpha, y MEP-2, péptidos muramil como los N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L- alanyl-Disoglutaminyl- L-alanine-2-( 1'-2'-dipalmitoyl-s- n-glycero-3 huydroxyphosphoryloxy)ethylamine (MTP-PE) etc; adjuvants derived from the family of CpG molecules, CpG dinucleótidos and synthetic oligonucleótidos that include CpG motifs, lysosum exoenzyme of C.Limosum and synthetic adjuvants such as PCPP, cholera toxin, Salmonella toxin, alum and the like, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 12% squalene / Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants, QuilA, microvesicles and exosomes.

[0071] The term "pharmaceutically acceptable" means that the vehicle or excipient must allow the activity of the compounds of the pharmaceutical composition, particularly the particle of the invention, that is, it must be compatible with said components, so that it does not cause harm to the organisms to which it is administered.

[0072] In a particular embodiment, the composition of the invention, in particular the pharmaceutical composition of the invention, further comprises a bioactive agent.

[0073] Such compositions and / or formulations thereof may be administered to an animal, including a mammal and thus a human, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intraventricular, intraarticular, intratumoral, oral, enteral, parenteral, intranasal, ocular or topically. A preferred route of administration of the compositions and / or formulations of the compound of the invention for the prevention and treatment of pain in a subject is the parenteral route. In a particular embodiment, the composition of the invention for use in the prevention and / or treatment of pain in a subject is formulated for administration topically, systemically (oral, nasal, sublingual, intravenous, intramuscular, subcutaneous, etc.) or by administration into the nervous system by nerve block.

[0074] In the present invention, “subject” means any animal, preferably a mammal, more preferably a primate, in particular a human being, of any race, sex or age.

[0075] In the present invention, "treatment" refers to the set of means used to decrease, lessen, alleviate, or eliminate pain. The International Association for the Study of Pain (IASP) defines pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage." Pain is, in principle, a defense mechanism, whose function is to detect and localize processes that damage bodily structures, but it is also a subjective phenomenon that may or may not be linked to an organic injury or pathology. Pain has a sensation component, often described as a process that penetrates or destroys tissue (piercing, oppressive), and an emotional component (anxiety, excitement, fear), influenced by physical, psychological, and social aspects, which frequently complicates its therapeutic approach.

[0076] Pain can be classified according to its duration: acute or chronic; according to its origin: nociceptive, neuropathic, and psychogenic; and / or according to its location: somatic, visceral, central, or peripheral. Any of these pains can be prevented and / or treated with the present invention.

[0077] Invention Kit

[0078] Administration of the peptide, polynucleotide, gene construct, vector, cell or pharmaceutical composition of the invention may require a set of components, which in turn may be arranged together in the form of a kit.

[0079] Thus, in another aspect, the invention relates to a kit, hereinafter the “kit of the invention”, comprising the peptide, polynucleotide, gene construct, vector, nanoparticle or composition of the invention, for use in the prevention and / or treatment of pain in a subject.

[0080] Useful components for its administration that may be included in the kit include, but are not limited to, buffer solution, lysis solution, sterile materials (such as syringes, swabs, or forceps), distilled water, or alcohols (ethanol). Additionally, the kit may contain instructions or guidelines to guide the person skilled in the art in its administration.

[0081] The terms used to define the kit and its use in the prevention and / or treatment of pain have already been explained, and both they and their preferred embodiments are applicable to the use of the kit of the invention.

[0082] Treatment / prevention method of the invention

[0083] In another aspect, the invention relates to a method for the treatment and / or prevention of pain in a subject, comprising administering to said subject the peptide, polynucleotide, gene construct, vector, nanoparticle, or composition of the invention.

[0084] The terms defined and explained for the remaining aspects of the invention, as well as the preferred embodiments thereof, are also applicable to the method of treatment and / or prevention of the invention.

[0085] DESCRIPTION OF THE FIGURES

[0086] Figure 1: Evolution of (A) blood glucose (mg / dL) and (B) weight (g) over 21 days in control rats (1 mL / kg saline solution, sc; normoglycemic; n = 10) or treated with alloxan (150 mg / kg, sc; diabetic; n = 38). p < 0.05 vs. normoglycemic.

[0087] Figure 2: Evolution of tail-flick time (s) at different measurement times (min) using the heat source method in normoglycemic (n=10) and diabetic (n=10) rats. P<0.05 vs. normoglycemic rats.

[0088] Figure 3: Evolution of tail wagging time (s) at different measurement times (min) using the heat source method in control diabetic rats (physiological saline, 1 mL / Kg, sc; n=10), or after subcutaneous administration of 10 mg / kg of morphine (diabetic morphine; n=8), 4 nmoles / g of peptide T AT- 0x43266-283 (diabetic peptide 4; n=10) or 8 nmoles / g of peptide TAT-Cx43266-283 (diabetic peptide 8; n=10). p<0.05 vs control diabetics. EXAMPLES

[0089] The invention will then be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the invention.

[0090] 1. Materials and methods

[0091] 1.1. Induction and maintenance of diabetes

[0092] Experiments were carried out on male Wistar rats (n=48) at the Animal Experimentation Service (SEA) of the University of Salamanca (USAL). The animals were maintained on a 12 / 12 hour light / dark cycle and housed in a special room at a constant temperature (22 ± 2°C) and humidity (50%) in the Animal Facility of the Faculty of Pharmacy at USAL (PAE-SA001; Salamanca, Spain). All animal procedures were approved by the corresponding Research Ethics Committee of USAL and by the General Directorate of Agricultural and Livestock Production of the Regional Government of Castilla y León (protocol no. 877), taking into account that the experiments in this project were carried out in accordance with: (i) the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (USA); (i) the ARRIVE guidelines for reporting animal experiments (Percie du Sert et al., 2020) and (i¡¡) the principles set out by European regulations (2010 / 63 / EU) and related Spanish legislation (RD 53 / 2013 and RD 1090 / 2015).

[0093] Diabetes induction was performed by subcutaneous administration of alloxan (150 mg / kg) (García et al., 2005, Br J Pharmacol, 145:593-601 ; García-Pedraza et al., 2022; Life Sci, 293:120335; Férnandez-González et al., 2022, Biomed Pharmacother, 153:113276); diabetic animals were maintained for 21 days. Two days after administration, blood glucose levels were monitored to assess hyperglycemia, a measurement that was performed once a week for the duration of treatment (García et al., 2005, Br J Pharmacol, 145:593-601; García-Pedraza et al., 2022; Life Sci, 293:120335; Férnandez-González et al., 2022, Biomed Pharmacother, 153:113276). Thus, an insulin treatment protocol was established that ensured the survival of the animals, maintaining the rats at blood glucose levels of around 500 mg / dl, that is, ensuring that, at the time of carrying out the experiment, the rats were diabetic.

[0094] Blood glucose measurement on day 2 (confirmation of the diabetic state) was performed by extracting a drop of blood from the rats' tail (using povidone as an antiseptic on the tail after extraction to avoid possible infections) and analyzing it with test strips for glucose determination using the Accuchek Aviva device (Roche), considering the animals hyperglycemic when the values ​​were higher than 300 mg / dl.

[0095] Subsequently, on days 7, 14, and 21 (once weekly), weight and blood glucose measurements were taken as described for day 2. Subsequent studies, in all cases, were carried out 3 weeks (21 days) after treatment with alloxan. In parallel, work was carried out in all cases with the corresponding control group of non-diabetic rats, which were injected subcutaneously with the same volume of physiological saline (alloxan vehicle). Throughout the housing period, the responsible researchers performed visual examinations, monitoring the general condition of the animals.

[0096] From the second day post-alloxan administration, all animals that had glucose levels below 300 mg / dl were excluded from the experiment.

[0097] Maintaining diabetic animals for three weeks allowed them to be used as an experimental model for the study of diabetic neuropathic pain.

[0098] 1.2. Distribution of experimental groups for the evaluation of neuropathic pain and analgesic activity using the heat focus technique.

[0099] After three weeks of confinement, the study of analgesic activity began. The heat focus technique on the rat's tail was used to evaluate pain (and the analgesic activity of the drugs being tested). In this protocol, the animal is placed in a clamp, and the tail is prepared for placement in a channel located below the heat focus. The tail is then illuminated with the heat focus at a voltage of 70–80 V (Kawakita K et al., 1987 Physiol Behav, 39:235–240), so that the light beam focuses on an area 2–4 ​​mm from the distal and dorsal part of the animal's tail. Tail movement latency is considered to be the time interval between the onset of radiant heat stimulation and the abrupt withdrawal of the tail from the nociceptive stimulus, taking this time as the measurement.Each rat was observed individually, and its tail was left exposed to radiant heat for a maximum of 10 seconds to avoid organ damage. To evaluate the analgesic activity of the TAT-Cx43266-283 peptide in induced pain, the animals were randomly divided into two groups, and the different compounds were administered subcutaneously (sc):

[0100] 1.- Normoglycemic group (n=10): control for all groups (administration of 1 mL / kg sc physiological saline)

[0101] 2.- Diabetic control group (n=10): evaluation of neuropathic pain (administration of 1 mL / kg sc saline solution)

[0102] 3.- Diabetic group - treatment with morphine (10 mg / kg, sc) (n=8): known analgesic that acts at the CNS level, prototype of a narcotic analgesic drug.

[0103] 4.- Diabetic group - treatment with TAT-Cx43266-283 (dose: 4 nmol / g, sc) (n=10)

[0104] 5.- Diabetic group - treatment with TAT-Cx43266-283 (dose: 8 nmol / g, sc) (n=10)

[0105] To carry out the experiment, measurements were taken at at=0 (prior to the administration of saline solution or drugs at different doses - see groups in this same section) and at at= 30 minutes, 90 minutes, 150 minutes and 210 minutes post-administration.

[0106] 1.3 Drugs used

[0107] The drugs used in these experiments were: alloxan (Merck, Spain), morphine (B. Braun Medical SA, Spain), and T AT- 0x43266-283 (SEQ ID NO: 3: AYFNGCSSPTAPLSPMSP). All of them were dissolved in saline solution (0.9% NaCl) or distilled water at the time of administration.

[0108] 1.4 Statistical treatment

[0109] Results are presented as mean ± SEM across 8–10 experiments. Student t-tests and one-way ANOVA were used to compare data between groups, with differences considered significant when p<0.05.

[0110] 2. Results

[0111] 2.1 Evolution of weight and blood glucose

[0112] Regarding the results obtained, the animals were diabetic from day 2 after alloxan administration and maintained stable hyperglycemia levels throughout their 21 days in the stable, while their normoglycemic controls had blood glucose levels of 110–120 mg / dl (Figure 1A). Similarly, periodic weight monitoring indicated that the diabetic animals did not gain weight as much as the control animals (Figure 1B).

[0113] 2.2 Hyperalgesia related to diabetic neuropathy

[0114] Regarding the hyperalgesia associated with diabetic neuropathy described for this experimental model, we can see that it is clearly visible in the heat focus test at all measurement times. If we compare diabetic animals with normoglycemic animals, we see that diabetic animals tend to withdraw their tails sooner than normoglycemic animals, with the withdrawal time being significantly shorter at all measurement times (Figure 2), confirming the hyperalgesia these animals suffer from due to diabetic neuropathy.

[0115] 2.3 Analgesic activity of T AT -0x43266-283 in the heat focus pain induction technique

[0116] With these preliminary considerations in mind, the results obtained show that diabetic rats treated with morphine (control drug) do not perceive pain and therefore do not tend to withdraw their tails from the heat source. This is why, at the measurement time of 30 minutes, the average reaches the maximum permitted exposure time to the source (10 s). As time progresses, the morphine is metabolized and the animals respond again by withdrawing their tails more quickly from the heat source (Figure 3).

[0117] Regarding the peptide, we found that analgesic activity began to appear at a dose of 4 nmol / g. The withdrawal time was significantly longer (compared to the diabetic control group) at the 8 nmol / g dose at all measurement times, remaining at its maximum for up to 150 min (unlike morphine, which begins to decline in analgesic effect after 90 min) (Figure 3).

[0118] 3. Conclusions

[0119] 1. The Src inhibitory peptide, TAT-Cx43266-283, exerts an analgesic effect on neuropathic pain determined by the heat focus method in a diabetic rat model of neuropathic pain. 2. The analgesic effect of TAT-Cx43266-283 is dose-dependent and reaches a maximum at 8 nmol / g after subcutaneous administration.

Claims

CLAIMS 1. A peptide comprising the amino acid sequence SEQ ID NO: 1 for use in the prevention and / or treatment of pain in a subject.

2. Peptide for use according to claim 1, comprising the amino acid sequence SEQ ID NO:

4.

3. Peptide for use according to claim 1 or 2, comprising the amino acid sequence SEQ ID NO:

5.

4. Peptide for use according to any one of claims 1 to 3, comprising the amino acid sequence SEQ ID NO:

6.

5. Peptide for use according to any one of claims 1 to 4, comprising the amino acid sequence SEQ ID NO:

3.

6. Peptide for use according to any one of claims 1 to 5, wherein the peptide has a length of 10 to 100 amino acids.

7. Peptide for use according to any one of claims 1 to 6, wherein the peptide is covalently linked to a cellular internalization amino acid sequence.

8. Peptide for use according to claim 7, wherein the cellular internalization amino acid sequence is attached to the amino terminal end of the peptide.

9. Peptide for use according to claim 7 or 8, wherein the cellular internalization sequence comprises the amino acid sequence SEQ ID NO:

7.

10. Peptide for use according to any one of claims 1 to 9, wherein the peptide comprises the amino acid sequence SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

26.

11. Peptide for use according to any one of claims 1 to 10, wherein the peptide is covalently linked to biotin.

12. A polynucleotide encoding a peptide according to any of the claims of claims 1 to 11 for use in the prevention and / or treatment of pain in a subject.

13. A gene construct comprising a polynucleotide according to claim 12 for use in the prevention and / or treatment of pain in a subject.

14. A vector comprising a polynucleotide according to claim 12, or a gene construct according to claim 13, for use in the prevention and / or treatment of pain in a subject.

15. Vector for use according to claim 14, wherein the vector is a viral vector, preferably a viral vector of a retrovirus, a lentivirus or an adenovirus.

16. A nanoparticle comprising a peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a gene construct according to claim 13 or a vector according to claim 14 or 15, for use in the prevention and / or treatment of pain in a subject.

17. Composition comprising the peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a gene construct according to claim 13, a vector according to claim 14 or 15, or a nanoparticle according to claim 16, for use in the prevention and / or treatment of pain in a subject.

18. Composition for use according to claim 17, further comprising a carrier and / or a bioactive agent 19. Composition for use according to claim 17 or 18, wherein the composition is formulated for topical, local, systemic or nervous system-directed administration.

20. Composition for use according to any one of claims 17 to 19, wherein the composition is a pharmaceutical composition.

21. A kit comprising a composition according to any one of claims 17 to 20, a peptide according to any one of claims 1 to 11, a polynucleotide according to claim 12, a gene construct according to claim 13, a vector according to claim 14 or 15, or a nanoparticle according to claim 16, for use in the prevention and / or treatment of pain in a subject.