WO2024031163A1 - Use of peptides and formulation - Google Patents

Abstract

The present invention describes opioid peptides that can be used to prepare antinociceptive medicines. Specifically, the present invention comprises three peptides with surprising antinociceptive efficacy and no detectable adverse effects. The present invention pertains to the fields of medicine, biochemistry and biophysics.

Description

Invention Patent Descriptive Report USE OF PEPTIDES AND FORMULATION Field of the Invention

[0001] The present invention describes opioid peptides that can be used to prepare antinociceptive medications. Specifically, the present invention comprises three peptides with surprising antinociceptive efficacy, and which do not present any detectable adverse effects. The present invention lies in the fields of Medicine, Biochemistry and Biophysics.

Background of the Invention

[0002] In the Portuguese language, the word pain originates from the Latin dolore, which means suffering, while the homologous pain, in the English language, derives from the Greek poiné, which refers to the concept of penalty (FEIN et al., 2011). Since the beginning of humanity, pain has accompanied man and, once disabling, and arising from various diseases, its control becomes one of the most important therapeutic priorities (TEIXEIRA; OKADA, 2009).

[0003] The concept of pain is complex because it is something subjective and personal. According to the International Association for the Study of Pain (IASP), pain is an unpleasant sensory and emotional experience associated, or similar to that associated, with real or potential tissue damage (RAJA et al., 2020).

[0004] Pain has also been described as a signaling from the Central Nervous System (CNS) to warn of possible tissue injuries, stimulating a series of adaptation and reflex reactions, aiming to distance the organism from the aggressor agent and preserve it (VITOR et al. al., 2008), and can therefore be considered as a protective function, in addition to being the only or most important symptom for the diagnosis of various diseases (DE OLIVEIRA; AKISSUE, 2009).

[0005] There are several ways to categorize pain and, among these, a possible classification, related to its duration, would be acute and chronic. Acute pain, which occurs due to the stimulation of nociceptors in response to tissue damage, is self-limiting and manifests for shorter periods (less six months) and generally with easily identifiable causes. Chronic pain can also originate from illness or injury, is persistent, constant and/or intermittent, almost always associated with chronic diseases, and is defined as pain that lasts for more than six months (MOORE, 2009; PORTH; GROSSMAN, 2013).

[0006] Taking into account its origin, pain can be classified as nociceptive (physiological), neuropathic (pathophysiological) or mixed. Nociceptive pain is a reflection of the good functioning of nociceptors that lead to the perception of harmful stimuli as being painful. Neuropathic pain is pathological, resulting from abnormal sensory processes as a result of injury to the nervous system. And in mixed pain there is a lack of definition in the components of the two types of pain (SMELTZER et al., 2012).

[0007] Thousands of people live with pain every day, being the most common reason for seeking medical care, disabling and generating anguish in more people than any single disease (SMELTZER et al., 2012). Among the ten main causes of death in the world are heart disease, stroke, respiratory problems and cancer, and it can be observed that pain is a characteristic present among the main causes of death in the world. Thus, pain can be considered one of the biggest clinical and economic problems in the world, a global health problem that brings negative impacts on the lives of those who live with it (ANDRÉ et al., 2019; ISMAIEL; DUMITRAÇCU, 2019; TORIÓ et al ., 2019).

[0008] Pain modulation is mainly regulated through the activation of the three classic types of opioid receptors, which are the p (mu or mi), K (kappa) and õ (delta) receptors expressed in neurons of the central and peripheral nervous system . Upon reaching the CNS, opioid receptors can be activated, enabling analgesia induced by endogenous or exogenous opioids. Where an opioid agonist would act by stimulating descending inhibitory pathways through the homeostasis and pain mediation center, the raphe magna nucleus (NRM), acting on the periaqueductal mass (PAG) and nucleus reticular paragigantocellularis (NRPG) and having an effect on inhibitors of activation of descending neurons (PATHAN; WILLIAMS, 2012).

[0009] The mechanisms related to the perception and processing of pain consist of: 1) Transduction: painful impulse is received by nociceptors and transformed into action potential; 2) Transmission: propagation of the nervous impulse to the central nervous system; 3) Modulation: before reaching higher levels, the impulse is modulated in the spinal cord; 4) Perception: the impulse is integrated and perceived as pain (DA FONSECA; FRIGO, 2018).

[0010] Although opiates and opioids are terms often used as synonyms, they have different meanings, with opiates being substances obtained from opium, having the base structure of morphine or thebaine and affinity with opioid receptors, while opioid refers to to any endogenous or exogenous substance that, despite not having the base structure of morphine or thebaine, has an affinity for opioid receptors (FORGET, 2019).

[0011] Among the main representatives of opioid drugs is morphine, which is considered the "gold standard" treatment for chronic and intense pain (DEVEREAUX; MERCER; CUNNINGHAM, 2018). Morphine is an organic alkaloid compound, a phenantrenic derivative with two planar rings and two aliphatic ring structures that occupy a plane approximately at right angles to the rest of the molecule (RANG et al., 2015), has a molecular formula of C17H19NO3 and a molar mass of 285.34 g/mol (STAUFFER; DANTAS; PRONKO, 2018). Its pharmacophoric group is N-methyl-y-phenylpiperidine, composed of a tertiary amino group that must be separated from an aromatic ring by two atoms and a quaternary carbon atom linked to a phenyl ring. The free hydroxyl groups in the benzene ring, linked to the nitrogen atom by two carbons, give the molecule opioid activity (FERREIRA; FACCIONE, 2005).

[0012] Morphine is an agonist drug, as its interaction with opioid receptors is capable of activating them, provoking a biological response. Although exerts its effects through interaction with p, õ and K receptors, it has greater affinity for p receptors (ROECKEL et al., 2017). Its main routes of administration are oral and intravenous, with the Central Nervous System as the target organ. The main route of metabolization of morphine is its conjugation with glucuronic acid, through hydroxyl groups and the two main metabolites formed are morphine-3-glucuronide and morphine-6-glucuronide. The half-life of morphine is approximately two hours and its elimination is mainly via the kidneys, through glomerular filtration (KATZUNG; TREVOR, 2017; WHALEN; FINKEL; PANAVELIL, 2016).

[0013] The three main groups of opioid receptors identified in humans and which are widely distributed throughout the body are: opioid peptide receptor p, OP3, MOP (mu or mi), found in the brain stem and thalamus; opioid peptide receptor K, OP2, KOP (kappa), located in the spinal cord, brain stem and limbic system; and peptide receptor õ, OP1, DOR (delta), located in the brain (WIFFEN et al., 2017; YE et al., 2012). Structurally, they are composed of seven transmembrane loops coupled to G proteins, each receptor has a highly conserved fold in its structure, called ECL2 (THOMPSON et al., 2012). Although they all have great structural similarity, they have different endogenous ligands, which can result in similar or distinct actions (AZZAM; MCDONALD; LAMBERT, 2019).

[0014] Opioids with characteristics similar to morphine can be considered full p-receptor agonists such as codeine, methadone, meperidine, fentanyl and tramadol, a group that does not have its activity limited by the ceiling effect, not having a maximum dose from which it is not possible to obtain greater analgesia. Medications such as buprenorphine are partial p-receptor agonists that have a ceiling effect. The group of mixed agonists/antagonists, represented by medications such as pentazocine, depending on the circumstances, presents characteristics agonists or antagonists, but their use is limited by high adverse effects (GOZZANI, 2020).

[0015] Pain control remains a complex and difficult task since both opioid and non-opioid analgesics do not provide universal pain relief or are free from harmful effects. Although opioids are efficient medications in controlling moderate to severe pain, their use requires great caution, as they are medications that have several side effects, such as nausea, dizziness, respiratory depression, itching, euphoria, dysphoria, sedation and, when used, for long periods, lead to the development of tolerance and dependence (BARFIELD et al., 2013; BARON; BINDER; WASNER, 2010; VOLKOW et al., 2014). Thus, specific approaches to opioid analgesia offer an interesting alternative and, consequently, lead to the discovery of new drugs for the treatment of pain that minimize, or nullify, the typical adverse effects of current opioids (MACHADO-VIEIRA; ZARATE JR, 2011).

[0016] Opioid peptides were identified for the first time in 1975, and since then, several researches have been dedicated to the search and development of new peptides with analgesic action and that do not present the same side effects as morphine and other analgesics (HUGHES et al., 1975). They are molecules that have diverse biological activity and interact with the p, K and õ opioid receptors (SZYMASZKIEWICZ et al., 2019).

[0017] Based on their origin, opioid peptides can be classified as exogenous or endogenous. Endogenous opioid pathways modulate pain in conditions of stress and/or survival risk, being composed of opioid neuropeptides and their receptors (PASTERNAK; PAN, 2013). Endorphins, whose main representatives are enkephalins, dynorphins, endorphins and endomorphins, are produced naturally in the body, initially they were identified in the CNS and later in various tissues and organs, including cells of the immune system (PEPE et al., 2004; PLEIN; RITTNER, 2018; SALZET, 2001). These peptides, also called typical opioid peptides, derive from the cleavage of precursor proteins (pro-enkephalin, pro-dynorphin and pro-opiomelanocortin) (JANECKA; FICHNA; JANECKI, 2004) and retain a YGG and YPF sequence in their N-terminus. , representing a typical sequence with a structure similar to morphine (ERDEI et al., 2018).

[0018] Exogenous peptides were discovered in the 1970s in foods and were called exorphins. These peptides of exogenous origin are also known as atypical peptides, as they carry several amino acids in their N-terminus with the conservation of tyrosine residues, being hydrolyzed from animal or vegetable proteins, a typical example of encryption (GARG; NURGALI; KUMAR MISHRA, 2016).

[0019] In the search for new drugs for pain control, in silico prospecting for encrypted peptides can be a promising source of opioid peptides that are often inactive when internalized into a larger protein sequence. This technique has been used successfully in the search for antimicrobials (BRAND et al., 2019; RAMADA et al., 2017; HINCAPIÉ; GIRALDO; ORDUZ, 2018) and has become a focus in the search for peptides with the most different actions such as hypotensive, anti-inflammatory, healing and analgesic (DOS ANJOS et al., 2019; HE et al., 2019; SUÁREZ et al., 2020; ZHOU et al., 2020), but is considered scarce when the approach is aimed at the screening of opioid peptides (VINECKY et al, 2015; GARG et al., 2018).

[0020] In the search for the state of the art in scientific and patent literature, the following documents were found that deal with the topic:

[0021] The document “Vinecky, Felipe. "Identification and characterization of opioid peptides present in the protein fraction of Coffea arabica grains." (2015).” reveals opioid peptides encrypted in Coffea arabica beans, identified through analysis of the coffee genome, available in international databases. The search was carried out focusing on identifying peptides that presented the N-terminal YGG region. The document does not disclose the peptides selected in the present patent application.

[0022] BR102014020352 is a patent document and discloses opioid peptides, comprising 7 amino acids and the N-terminal YGG region, with the fourth and fifth amino acids being nonpolar, and pharmaceutical and food compositions comprising said peptides. Furthermore, this document discloses the use of said compositions to promote analgesia and/or satiety to an individual. Furthermore, the document reveals that peptides with an N-terminal region of the YP type could be casomorphins, morphiceptins, hemomorphins or endomorphins, in addition to revealing that the majority of biologically active peptides would be encrypted in animal or vegetable proteins. Although this document generally reveals opioid peptides, there is great variability between the peptides, therefore, even peptides that fit the markush formula of this document do not necessarily have opioid action. Furthermore, this document does not explicitly disclose the peptides of the present patent application, nor does it suggest their surprising potential as an antinociceptive agent.

[0023] Document US6303578B1 is a patent document and discloses linear or cyclic opioid peptides, capable of binding to p-receptors. The document reveals peptide sequences with N-terminal regions of the YPW type, with the amino acid in position 3 being aromatic and the amino acid in position 4 being nonpolar. Finally, the document reveals the use of said peptides in pharmaceutical formulations to promote analgesia, alleviate gastrointestinal diseases and chemical dependency treatments. This document does not disclose the peptides described in the present patent application.

[0024] Thus, from what can be inferred from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity compared to the state of the art.

[0025] Therefore, it is necessary to develop new assets effective antinociceptives with few adverse effects, both to overcome the development of tolerance and dependence by patients, and to improve the population's quality of life.

Summary of the Invention

[0026] In this way, the present invention solves the problems of the prior art using three surprisingly effective opioid peptides, which do not present any detectable adverse effects.

[0027] In a first object, the present invention presents the use of peptides in the preparation of opioid and/or antinociceptive medications, wherein the peptides are as defined in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO: 3.

[0028] In a second object, the present invention presents the use of peptides in the preparation of medicines for the treatment of pain, cough, diarrhea, shortness of breath and/or hyperalgesia, wherein the peptides are as defined in SEQ ID NO: 1 , SEQ ID NO:2 or SEQ ID NO:3.

[0029] In a third object, the present invention presents the use of peptides in the preparation of medicines to treat and/or control dependence and/or tolerance to opium and/or opioids.

[0030] In a fourth object, the present invention presents a formulation comprising at least one peptide as defined by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and at least one excipient.

[0031] These and other objects of the invention will be immediately valued by those skilled in the art and will be described in detail below.

Brief Description of Figures

[0032] The following figures are displayed:

[0033] Figure 1 shows a comparative molecular modeling image of the Met-Enk peptides (A), SEQ ID NO:1 (B), SEQ ID NO:2 (C), SEQ ID NO:3 (D ) and morphine-receptor (E) using the methionine-enkephalin peptide as a template structure (PDB code: 1 PLW). All structures presented were subjected to 5 ns of structural refinement through dynamics molecules in aqueous solution (ionic strength = 0.15 M NaCI). The three-dimensional structure for morphine was obtained from DrugBank (code: DB00295) and subjected to the same energy minimization and structural refinement steps described above.

[0034] Figure 2 shows a three-dimensional representation for the molecular complexes with the highest affinity in kcal.mol' ¹ for the peptide-receptor systems (Met-Enk (A), SEQ ID NO:1 (B), SEQ ID NO: 2 (C), and SEQ ID NO:3 (D)) and morphine-receptor (E). In gray sticks, amino acid residues in the p (mu) receptor (PDB: 5C1 M) involved in atomic interactions with peptide or morphine residues are represented.

[0035] Figure 3 shows a graph of the antinociception index at different times after application (i. p.) of the peptide of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in mice using the hot plate test for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001 between saline x morphine and saline x enkephalin.

[0036] Figure 4 shows a graph of the antinociception index at different times in the control groups in the hot plate test for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***saline x enkephalin+naloxone and ##saline x morphine+naloxone.

[0037] Figure 5 shows a graph of the antinociception index at different times after application (i. p.) of the peptide of SEQ ID NO:1 in mice using the hot plate test for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001 between Salina x SEQ ID NO:1.

[0038] Figure 6 shows a graph of the antinociception index at different times after application (ip) of the peptide of SEQ ID NO:2 in mice using the hot plate test for 240 minutes. Values represent the mean ± sem (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001 between Salina x SEQ ID NO:2.

[0039] Figure 7 shows a graph of the antinociception index at different times after application (i. p.) of the peptide of SEQ ID NO:3 in mice using the hot plate test for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001 between Salina x SEQ ID NO:3.

[0040] Figure 8 shows a graph of the antinociception index at different times after application (i. p.) of the peptide of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in mice using the blood withdrawal test. tail for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***saline x morphine and ## saline x enkephalin.

[0041] Figure 9 shows a graph of the antinociception index at different times in the control groups in the tail withdrawal test for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). *p<0.05 between saline x enkephalin+naloxone; ns between saline x morphine+naloxone.

[0042] Figure 10 shows an index of antinociception at different times after application (i.p.) of the peptide of SEQ ID NO:1 in mice using the tail flick test for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001 between Salina x SEQ ID NO:1.

[0043] Figure 11 shows an index of antinociception at different times after application (i.p.) of the peptide of SEQ ID NO:2 in mice using the tail flick test for 240 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001 between Salina x SEQ ID NO:2.

[0044] Figure 12 shows an index of antinociception at different times after application (ip) of the peptide of SEQ ID NO:3 in mice using the tail flick test for 240 minutes. The values represent the mean ± sem (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001 between Salina x SEQ ID NO:3.

[0045] Figure 13 shows the effect of administering POUCB's evaluated in the 5-minute open field test on BF, CP, CC, AL, LV and AM. Values represent the mean ± standard deviation (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001; **p<0.01; *p<0.05 in relation to the saline group; ### p<0.001; ##p<0.01 ; #p<0.05 in relation to the morphine group. BF: fecal cakes; CP: intersections on the outskirts; CC: intersections in the center; AL:self-cleaning; LVJevantamentos; AM: absence of movements.

[0046] Figure 14 shows a graph of the effects of the administration of saline, morphine, enkephalin and peptides in the elevated plus maze test for 5 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001; **p<0.01; *p<0.05 in relation to the saline group; ### p<0.001; ##p<0.01 ; #p<0.05 in relation to the morphine group. EBF: amount of entry into closed arms; EBA: amount of entry into open arms; TBF: time spent in closed arms; TBA: time spent in open arms; TC: length of stay at the center.

[0047] Figure 15 shows the effects of administering control treatments in the elevated plus maze test for 5 minutes. The average of the values represented in ± e.p.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001; **p<0.01; *p<0.05 in relation to the saline group; ### p<0.001; ##p<0.01; #p<0.05 in relation to the morphine group. EBF: amount of entry into closed arms; EBA: amount of entry into open arms; TBF: time spent in closed arms; TBA: time spent in open arms; TC: length of stay at the center.

[0048] Figure 16 shows the effects of administering SEQ ID NO:1 in the elevated plus maze test for 5 minutes. The mean values represented in ± sepm (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001;**p<0.01;*p<0.05 in relation to the group saline; ### p<0.001;##p<0.01;#p<0.05 in relation to the morphine group. EBF: amount of entry into closed arms; EBA: amount of entry into open arms; TBF: time spent in closed arms; TBA: time spent in open arms; TC: length of stay at the center.

[0049] Figure 17 shows the effects of administering SEQ ID NO:2 in the elevated plus maze test for 5 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001; **p<0.01; *p<0.05 in relation to the saline group; ### p<0.001; ##p<0.01; #p<0.05 in relation to the morphine group. EBF: amount of entry into closed arms; EBA: amount of entry into open arms; TBF: time spent in closed arms; TBA: time spent in open arms; TC: length of stay at the center.

[0050] Figure 18 shows the effects of administering SEQ ID NO:3 in the elevated plus maze test for 5 minutes. Values represent the mean ± s.e.m. (n=7 animals per group), (Two-way ANOVA followed by Bonferroni post-test). ***p<0.001; **p<0.01; *p<0.05 in relation to the saline group; ### p<0.001; ##p<0.01 ; #p<0.05 in relation to the morphine group. EBF: amount of entry into closed arms; EBA: amount of entry into open arms; TBF: time spent in closed arms; TBA: time spent in open arms; TC: length of stay at the center.

Detailed Description of the Invention

[0051] The present invention describes opioid peptides encrypted in proteins present in the genome of autumn saffron or meadow saffron (Colchicum autumnale), pitanga (Eugenia uniflora) and papaya (Carica papaya), which were prospected in silico with based on a consensus sequence and physicochemical characteristics similar to other peptides that present opioid activity, chemically synthesized, purified and had their analgesic activities validated by in vivo tests, which were surprisingly high, without any detectable adverse effects.

[0052] The three peptides described in the invention were selected from a list of more than 100 possible opioid peptides listed from different genomes, based on consensus sequence and physicochemical parameters. These were selected, together with seven other peptides, to the detriment of all others, after carrying out docking with the p receptor, a test which indicated a possible greater interaction of these peptides with the opioid receptor. Of these ten peptides, four were synthesized and tested, with only three returning good results, which indicates a high degree of unpredictability in obtaining molecules with opioid activity, since other factors in the living organism can influence the activity.

[0053] The selected peptides demonstrated surprisingly high opioid analgesic potential, being defined by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and being effective active ingredients for new antinociceptive drugs and representing advances in the field of pharmacology and public health in general, since pain is still a public health problem, where thousands of people live with it daily, being the most common reason for seeking medical care, disabling and generating anguish in more people than any other isolated disease.

[0054] Although there are numerous analgesics available on the market, the most effective drugs today for controlling severe pain produce several side effects such as itching, euphoria and dysphoria, nausea, respiratory depression, dizziness and even dependence. Therefore, it is essential to search for new medication options that are as effective as current ones, but with fewer adverse effects.

[0055] Therefore, the peptides selected from this patent application, in addition to showing high efficacy as an antinociceptive active, also present fewer adverse effects than other already known drugs.

[0056] It is noteworthy that although the conserved sequence TyrGIyGly (YGG) and TyrProPhe (YPF) are present in enkephalin-like opioids and endomorphin-like opioids, respectively; the amino acids that follow after such a sequence directly influence the potential antinociceptive of peptides tested so far, as these can influence the ability of the pharmacophoric group to interact with the receptor.

[0057] In this sense, Vinecky (2015) demonstrated that some peptides with the conserved sequence TyrGIyGly did not show opioid activity (PSLEM11042 - YGGTGAH), even with residues compatible with that shown in document BR102014020352-4 A2.

[0058] In document BR102014020352-4 A2, the following markush sequence for opioid peptides was defined: TyrGlyGLy-X1 -X2-X3-X4, where X1 are uncharged or negatively charged polar amino acids at physiological pH (7.4) (Thr and Glu), X2 non-polar or slightly non-polar amino acids without charge (Ala, Gly or Thr), zero, considering the physiological pH of 7.4 (Gin, Thr or His).

[0059] The fact that a peptide synthesized by Vinecky (2015) presents the expected pattern of opioid sequences described in BR102014020352-4 A2, but does not present activity, proves that each peptide is a unique entity to be evaluated independently. The new peptides of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 do not have the same residue pattern or sequence for positions X1, X2, X3 and X4 described in document BR102014020352-4 A2; and even so, they demonstrated surprisingly high opioid analgesic potential.

[0060] In a first object, the present invention presents the use of peptides in the preparation of opioid and/or antinociceptive medications, wherein the peptides are as defined in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO: 3.

[0061] In a second object, the present invention presents the use of peptides in the preparation of medicines for the treatment of pain, cough, diarrhea, shortness of breath and hyperalgesia, wherein the peptides are as defined in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0062] In a third object, the present invention presents the use of peptides in the preparation of medicines to treat and/or control dependence and/or tolerance to opium and/or opioids.

[0063] In a fourth object, the present invention presents a formulation comprising at least one peptide as defined by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and at least one excipient.

[0064] In one embodiment, the medicine is used to treat diseases associated with pain selected from cancers, bacterial, viral, fungal and parasitic infections, autoimmune and/or inflammatory diseases, burns and trauma. In one embodiment, the medicament is used for the treatment and/or control of dependence and/or tolerance to opium and/or opioids.

[0065] In one embodiment, the medicine is applied intravenously, intrathecally or orally; preferably intravenous or intrathecal; even more preferably intravenous. In one embodiment, the formulation is applied intravenously, intrathecally or orally; preferably intravenous or intrathecal; even more preferably intravenous. In one embodiment, the formulation is in the form of a solution, suspension or emulsion; preferably solution or suspension. In one embodiment, the formulation is sterile.

[0066] In one embodiment, the formulation comprises an aqueous vehicle, preferably the aqueous vehicle comprises 0.15M NaCl or PBS buffer solution. In one embodiment, the formulation is in the form of tablets, capsules, or powder. In one embodiment, the formulation is a pharmaceutical formulation or medicament for treating pain, cough, diarrhea, shortness of breath and hyperalgesia. In one embodiment, the formulation is an opioid and/or antinociceptive medication.

[0067] In one embodiment, the concentration of peptides in pharmaceutical formulations and/or medicines is from 1 pM to 50 pM. In one embodiment, the concentration of the peptides is 10 pM to 40 pM. In one embodiment, the concentration of the peptides is 20 pM to 30 pM, the concentration of the peptides is 26 pM.

[0068] In one embodiment, the formulation additionally comprises a active compound selected from the group consisting of anticonvulsants, antidepressants, antipsychotics, anxiolytics, local anesthetics, corticosteroids, ketamines, non-steroidal anti-inflammatory drugs, muscle relaxants and antihypertensives.

[0069] In a fifth object, the present invention presents a method of treating pain or diseases associated with pain. In one embodiment, the method comprises the use of a formulation comprising at least one peptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or combinations thereof. In one embodiment, the formulation is an opioid and/or antinociceptive medication.

[0070] Definitions of:

[0071] Antinociceptive: refers to the ability to reduce or nullify the perception of pain. Opioid: refers to any endogenous or exogenous substance that, despite not having the base structure of morphine or thebaine, has an affinity for opioid receptors. Vehicle: refers to the medium in which the active ingredient is dispersed or solubilized in a formulation. Formulations

[0072] Liquid formulations can be in the form of solutions, suspensions or emulsions, preferably in the form of sterile solutions or suspensions that can be applied intravenously or intrathecally.

[0073] Preferably, the vehicle used in liquid formulations of the present patent application is aqueous, wherein preferably the aqueous vehicle comprises 0.15M NaCl or buffer solution pH 7.3-7.5, such as PBS buffer. Preferably, the aqueous vehicle comprises 0.15 M NaCl. Solid and/or semisolid formulations may be in tablet, capsule or powder form.

[0074] Excipient: refers to any compound in a formulation that is not the active principle or compound. Excipients in formulations are used to adjust different parameters, such as pH, viscosity, active solubility, formulation stability, homogenization, etc., yet, excipients are widely known in the state of the art, non-limiting examples that can be used in the context of the present patent application are listed below: Diluents: sugars, such as lactose, dextrin, glucose, sucrose, sorbitol; inorganic compounds such as silicates, calcium and magnesium salts, sodium and potassium chloride. Binders: natural or synthetic polymers, such as starch, sugars, alcohols and cellulose derivatives. Disintegrants: starch, cellulose derivatives, alginates, polyvinylpyrrolidone. Lubricants: stearic acid, such as magnesium stearate. Stabilizers: anionic, cationic, amphoteric and non-ionic surfactants; chelators, such as citric acid, EDTA. Preservative system: parabens (p-hydroxybenzoates).

[0075] Formulations may comprise additional active compounds that increase the pharmacological activity of opioids, non-limiting examples of classes of additional active compounds are: anticonvulsants, antidepressants, tricyclic antidepressants, antipsychotics, anxiolytics, local anesthetics, corticosteroids, ketamines, anti-inflammatories non-steroidal, muscle relaxant and antihypertensive drugs.

[0076] Additional active compounds may be selected from the group consisting of carbamazepine, phenytoin, amitriptyline, clomipramine, imipramine, lidocaine, mexiletine, paracetamol, metamizole, acetylsalicylic acid, indomethacin, diclofenac, ibuprofen, naproxen, piroxicam, meloxicam, mefenamic, ketorolac, baclofen and clonidine.

Diseases associated with pain

[0077] Due to pain being a response that can be caused by a variety of clinical conditions, or even have an idiopathic origin. The peptides of the present patent application defined by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 can be used to treat a variety of diseases associated with pain. Non-limiting examples are described below. Cancers: melanoma, lung cancer, cervical, prostate, colon and rectum, stomach, oral cavity, esophagus, bladder, larynx, leukemias, lymphomas, breast, thyroid, ovary, liver, pancreas, central nervous system. Infectious diseases bacterial, viral, fungal or parasitic: cholera, Chagas disease, typhoid fever, hepatitis A, hepatitis E, leptospirosis, poliomyelitis, toxoplasmosis, worms, schistosomiasis, rabies, tetanus, dengue fever, malaria, yellow fever, mumps, diphtheria, meningococcal disease, flu, pneumonia, severe acute respiratory syndrome, measles, rubella, tuberculosis, chickenpox, AIDS, hepatitis B, syphilis. Autoimmune and/or inflammatory diseases: lupus, arthritis, multiple sclerosis, Crohn's disease. Burns and trauma. Complications arising from diabetes and hypertension: limb necrosis, liver, kidney and heart failure, neuropathies, stroke, myocardial infarction.

Replacement therapy

[0078] Opioids were also possibly the first drugs of abusive recreational use, being used to this day for non-clinical purposes. It is known that opioids produce several side effects such as itching, euphoria and dysphoria, nausea, respiratory depression, dizziness, in addition to their prolonged use leading to dependence and tolerance. This presents a problem, where sudden withdrawal can cause withdrawal.

[0079] Due to the various adverse and side effects of the use of opium or opioids exemplified above, compounds that present mechanisms similar to opium or opioids widely used clinically or abused, but which have lower potency and/or a longer half-life and /or reduction of adverse effects may be presented as potential drugs for use in replacement therapies, thus reducing the adverse effects of withdrawing from the use of opium or opioids.

[0080] Thus, the encrypted peptides of the present patent application defined by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 can be used to prepare a medicine for the treatment and/or control of addiction and/ or tolerance to opium and/or opioids.

Examples

[0081] The examples shown here are intended only to exemplify a of the countless ways to carry out the invention, however without limiting its scope.

[0082] To prospect for the three peptides of the present invention, a list of plant species known for their antinociceptive activities was initially made. Then, the database of the National Center for Biotechnology Information (NCBInr) was used to search for sequences of possible encrypted natural opioid peptides for the classes: enkephalins and dynorphins, endorphins and endomorphins, with the choice of peptide sequences being made according to with the characteristics of their amino acid sequences, using the Notepad++ program as assistance.

[0083] After the prospecting process, the three peptides of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 were selected, which were chemically synthesized, evaluated by molecular modeling, refinement of structures by molecular dynamics, study of peptide/receptor interactions and had their analgesic activities validated by in vivo tests. The antinociceptive action of the three peptides was evaluated in vivo using classic methodologies for the analgesic evaluation of substances, such as the Hot Plate and Tail Flick tests. The peptides demonstrated analgesic potential similar to, and at certain times even superior to, morphine, used as a control in analgesia tests. Furthermore, all peptides had their effects blocked after administration of naloxone, a classic opioid antagonist, thus demonstrating the opioid characteristic of the peptides.

[0084] Thus, the three peptides described here can be used more effectively in the treatment of pain in men, consequently improving their quality of life. Details of the selection process for these peptides, with effective antinociceptive properties and low side effects, are described below.

[0085] Selection of Peptides, synthesis, characterization and purification

[0086] Firstly, a search was carried out in databases on plants previously described popularly or scientifically as having analgesic action. After selecting approximately 100 plant species, we began searching for their genomic sequences in the non-redundant database of the National Center for Biotechnology Information (NCBInr).

[0087] All plants that had their protein sequences published were analyzed, through a search for sequences of possible encrypted natural opioid peptides for the classes: enkephalins, dynorphins, endorphins and endomorphins (YGG, YGGF, YGGW, YPF, YPW), through the similarity of identity of N-terminal amino acids, selecting protein sequences with 7 to 11 amino acids with sequences, using the Notepad++ program as assistance.

[0088] Once the peptide sequences were chosen according to the characteristics of their amino acid sequences, the peptides SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 were selected and synthesized using a protocol of chemical synthesis.

[0089] The synthesis was developed according to the F-moc solid phase methodology (use of 9-fluorenylmethoxycarbonyl as protector of the amino group), purified (> 95%), lyophilized and stored. Verification of molecular masses, degree of purity and amino acid sequence of the synthesized peptides was carried out by ESI-Q-TOF Electrospray-Quadrupole-Time of flight Micro-TOF-Q III mass spectrometry, Bruker Daltonics®. Briefly, each peptide was mixed in a 1:1 acetonitrile:ultrapure water solution containing 0.1% (v/v) formic acid and injected directly into the equipment at a flow rate of 180 pL/h, using a nebulization pressure of 4 bar, flow of 4L/min of dry gas and 200 °C. Data were obtained using the oTOF-Control software using the Tune Wide method, in a range of 50-1500 m/z, followed by the selection of the monoisotopic mass of each peptide for fragmentation. Data analysis and obtaining the sequence of each peptide was performed using the Data Analysis software (Bruker Daltonics).

[0090] Thus, the peptides were characterized by mass spectrometry, confirming the expected mass of each of them (table 1), as well as the amino acid sequence (table 2).

Table 1. Characterization of Peptides by mass spectrometry.

Table 2. Peptides as potential opioids selected.

Identification Amino acid sequence

SEQ ID NO:3 (POUCB5) YGGLLDR

[0091] Molecular modeling

[0092] Comparative molecular modeling of the Met-enk peptides and SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 was carried out using the Modeller 9v20 software (ALTSCHUL et al., 1997). The nuclear magnetic resonance (NMR) structure for the methionine-enkephalin peptide (PDB code: 1 PLW) was used as a template. The method used to construct the three-dimensional models was modeling by satisfying spatial restrictions. In total, 100 three-dimensional models were generated for each study peptide. From these theoretical models, those with the lowest free energy (DOPE score) were selected for refinement and structural validation. The three-dimensional structures were visualized and analyzed using the PyMOL program (DeLANO, 2002).

Table 3. Structural validations based on the stereochemistry of the shortest peptides

^The z-score values obtained for all structures reported here are in agreement with values observed for peptides of similar size, structurally studied by nuclear magnetic resonance (NMR) and deposited in the Protein Data Bank (PDB). ^b MolProbity score combines the clashscore, rotamers, and Ramachandran map evaluations into a single score.

Table 4. Average binding affinities in kcal.mol ^-1 for the peptide-receptor and morphine-receptor molecular complexes after 50 individual molecular docking simulations. _

_ Met-Enk _ SEQ ID NQ:! SEQ ID NQ:2 SEQ ID NQ:3 Morphine

p (mu) receiver -7.4 -7.1 -6.5 -5.9 -6.5

(PDB: 5C1M)

[0093] Structural refinement by molecular dynamics

[0094] The refinement of the theoretical lowest free energy structures generated by comparative molecular modeling was carried out through molecular dynamics simulations in aqueous solution. The force field used was GROMOS96 43A1 and the dynamics were carried out using the GROMACS V.5 computational package (LINDAHL; HESS; VAN DER SPOEL, 2001). The theoretical models were first immersed, individually, in cubic boxes with optimized sides, followed by their solvation with water molecules with single charge points. Chlorine ions (Cl) were inserted to neutralize the charge of the system and the simulations were conducted under an ionic strength equal to 0.15 M NaCl. The geometry for the water molecules was conditioned using the SETTLE algorithm (MIYAMOTO; KOLLMAN, 1992). All atom bond lengths were linked by the LINCS algorithm (HESS; LINCS, 1997). Electrostatic corrections were made using the Particle Mesh Ewald (PME) algorithm, with a radius having a cut off of 1.4 nm, in order to minimize computational simulation time. The same cut off for the radius was also used for the van der Waals interactions. The list of neighbors for each atom was updated every 5,000 simulation steps of 2 fs each. The systems were subjected to 50,000 energy minimization steps using the steepest descent algorithm. Then, the systems were subjected to temperature (310 K) and pressure (1 bar) normalization. The systems with minimized energy, balanced temperature and pressure were subjected to 5 ns of molecular dynamics simulation using the leap-frog algorithm, aiming at the refinement and structural relaxation of the theoretical models. generated for further molecular docking analyses.

[0095] Study of peptide/membrane interactions (molecular docking)

[0096] The AUTODOCK 4.2 software (TROTT; OLSON, 2010) was used in order to predict the binding affinities and atomic interactions for the peptide-p receptor (mu) and morphine-p receptor (mu) molecular complexes. With the aid of the AutoDock Tools tool, all hydrogen atoms were added to the structures and a three-dimensional box of dimensions 40 x 40 x 30 Â3 with 1 Â spacing was centered on the extracellular surface of the crystallographic structure for the p (mu) receptor ( PDB: 5C1 M). For peptides, maximum freedom for side chains was unlocked, and blocked for the main chain. Fifty molecular docking simulations were performed and the generated structures were classified according to their affinity values in kcal.mol' ¹ . The PyMOL program (2) was used to characterize the peptide/receptor and morphine-receptor interactions respecting the distance of 3.6 Å for blind docking.

Table 5. Predicted atomic interactions for the p(mu) receptor - peptides and p(mu) receptor - morphine complexes

[0097] Assessment of the analgesic potential of peptides

[0098] To determine the antinociceptive activity of the peptides, in vivo biological tests were carried out at the UCB Multiassay Laboratory, following the guidelines of COBEA (Brazilian College of Animal Experimentation) and with approval provided by the Ethics Committee for the use of animals. experimentation at the Catholic University of Brasília (UCB) under protocol no. 02/2017.

[0099] To carry out the tests, male Mus musculus mice of the balb/c lineage, approximately 6-8 weeks old, weighing between 20-25 g, acquired from the UCB vivarium, were used. The animals remained in cages and were kept under controlled conditions of light (12-hour light/dark cycle), temperature (23 ^S C) and humidity (55%). Before the entire experimental period and throughout it, the animals received water and food ad libitum.

[0100] Division of animals into groups

[0101] To carry out the in vivo tests, we used three candidate peptides and eleven groups of animals (n=07). One group received only the dose of peptide equimolar to that used for morphine sulfate (10 mg. kg' ¹ of animal; 26.08 pM), and the other group, the peptide at the same previous concentration + naloxone, in the form of morphine hydrochloride. naloxone (0.4 mg.mL' ¹ ) (Narcan, Cristália, Brazil) at a dose of 1 mg. kg ^-1 (2.75 pM), an amount sufficient to cause antagonism. We had three control groups: 1 - positive control (n=07) that received morphine sulfate (10mg.kg ^-1 of animal; 26.08 pM); 2 - negative control (n=07) that received a sodium chloride solution (0.9%, w/v): 3 - antagonism control (n=07) that also received morphine (10 mg. kg ^-1 of animal; 26.08 pM) + naloxone (1 mg. kg ^-1 , 2.75 pM). All administrations are made with a total volume of 100 ml intraperitoneally (ip).

[0102] Group E1 (composed of 7 animals): Intraperitoneal administration of Enkephalin at the concentration stipulated by the dose and molarity of morphine (10 mg. kg ^-1 of animal; 26.08 pM). Group E2 (composed of 7 animals): Intraperitoneal administration of peptide of SEQ ID NO:1 at the concentration stipulated by the dose and molarity of morphine (10 mg. kg ¹ of animal; 26.08 pM). Group E3 (composed of 7 animals): Intraperitoneal administration of peptide of SEQ ID NO:2 at the concentration stipulated by the dose and molarity of morphine (10 mg. kg ^-1 of animal; 26.08 pM). Group E4 (composed of 7 animals): Intraperitoneal administration of peptide of SEQ ID NO:3 at the concentration stipulated by the dose and molarity of morphine (10 mg. kg ¹ of animal; 26.08 pM). Group E5 (composed of 7 animals): Intraperitoneal administration of Enkephalin + Naloxone in the form of naloxone hydrochloride (0.4 mg.mL' ¹ ) (Narcan, Cristália, Brazil) at a dose of 1 mg. kg ^-1 (2.75 pM). Group E6 (composed of 7 animals): Intraperitoneal administration of peptide from SEQ ID NO:1 + Naloxone in the form of naloxone hydrochloride (0.4 mg.mL' ¹ ) (Narcan, Cristália, Brazil) at a dose of 1 mg. kg' ¹ (2.75 pM). Group E7 (composed of 7 animals): Intraperitoneal administration of peptide from SEQ ID NO:2 + Naloxone in the form of naloxone hydrochloride (0.4 mg.mL' ¹ ) (Narcan, Cristália, Brazil) at a dose of 1 mg. kg' ¹ (2.75 pM). Group E8 (composed of 7 animals): Intraperitoneal administration of peptide from SEQ ID NO:3 + Naloxone in the form of naloxone hydrochloride (0.4 mg.mL' ¹ ) (Narcan, Cristália, Brazil) at a dose of 1 mg. kg' ¹ (2.75 pM).

[0103] Control groups: Control C1 (consisting of 7 animals): Administration intraperitoneal morphine sulfate (10 mg. kg ^-1 of animal; 26.08 pM). Control C2 (consisting of 7 animals): Intraperitoneal administration of morphine sulfate (10 mg. kg ^-1 of animal; 26.08 pM) + Naloxone in the form of naloxone hydrochloride (0.4 mg.mL' ¹ ) (Narcan , Cristália, Brazil) at a dose of 1 mg. kg' ¹ (2.75 pM). Control C3 (consisting of 7 animals): Intraperitoneal administration of physiological sodium chloride solution, saline (0.9%, w/v).

[0104] Antinociception tests

[0105] To evaluate a possible antinociceptive activity of the peptides, we used the hot plate test and the tail flick test. Both are classic models of analgesia, where antinociception is expressed by comparing the latency, which is the response time from the induced thermal stimulus, in which the mean basal latency (measured before starting administration), the test latency (obtained after administration) and by maximum latency (stipulated to minimize possible injuries in groups of mice). It is best exemplified in the following formula (I):

AI = (Test latency - Basal latency)

(Max latency - Basal latency)

[0106] Hot plate test (WOOLFE & MACDONALD, 1944)

[0107] To perform the hot plate test, the mouse was placed on a plate heated to approximately 52 ^S C, and the time spent on the equipment was recorded until they showed the reaction of lifting or licking their paws, which are brain responses. considered integrated supraspinal responses.

[0108] After intervals of 30, 60, 90,120, 150, 180, 210 and 240 minutes of application of treatments, each animal was placed in contact with the hot plate and time measurement was stopped when the animal showed one of the signs above described. In order to prevent tissue damage, a maximum time of 30 seconds was established for the animals to be removed from the heated plate. For these tests, the animals were previously selected, where only the animals that showed a response within 30s were selected.

[0109] When evaluating the results presented in the hot plate test in Figure 3, we can observe that the saline group presented statistically significant values p<0.001 when compared with the morphine and enkephalin groups. In figure 4 we continue to find the characteristic pattern of the control groups when we compare saline with morphine+naloxone and saline with enkephalin+naloxone.

[0110] In Figures 5, 6 and 7 we can observe that all three peptides selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 presented statistically significant values in relation to saline, demonstrating an analgesic effect, We can also observe that at certain times they maintained an action similar to morphine and blockage of action when receiving noloxone together with the peptide, a typical characteristic of opioid analgesics.

[0111] When evaluating the results of the SEQ ID NO:1 peptide in the hot plate test (Figure 5), we can observe that Morphine only presented a higher and statistically significant value in relation to SEQ ID NO:1 at times 30 and 90. And SEQ ID NO:1, when compared with the SEQ ID NO:1 + naloxone group, presented higher and statistically significant values at times 30, 60 and 90 minutes, times when the SEQ ID NO:1 + naloxone peptide group when compared to the saline group, there was no significant difference, demonstrating that there was a blockade of the analgesic action during this period due to the use of naloxone, data that corroborates the literature, since naloxone has a half-life of 43 to 90 minutes and a duration of action of approximately 1.5 hours.

[0112] With the response presented by the groups in the evaluation of the peptide of SEQ ID NO:2, we can observe that again morphine only presented higher and statistically significant values in relation to SEQ ID NO:2 at times 30 and 60. When compared to group SEQ ID NO:2, the group SEQ ID NO:2 + naloxone also only presented lower values statistically significant in the first two test times (Figure 6).

[0113] In Figure 7, Morphine only presents statistically significant values in relation to the peptide of SEQ ID NO:3 in the first test time, and the SEQ ID NO:3+nalone group only maintained lower and statistically significant values in relation to SEQ ID NO:3 in times 30, 60 and 90.

[0114] Latency test for the tail withdrawal reflex (D'AMOUR AND SMITH, 1941)

[0115] Aiming to expand the characterization of the antinociceptive profile, we performed the tail-flick test. To do this, the mice were placed on equipment called a digital analgesimeter, so that their tails were in contact with the resistance capable of increasing the temperature.

[0116] When the animal stopped moving, the resistance was activated together with a timer and, when the animal made any movement with its tail, a sensor on the equipment itself detected and recorded the time the tail spent on the resistance and also the temperature that the animal withstood before moving its tail. The thermal stimulus causes the mouse's tail to be removed, through a reflex of spinal origin (BERGE, 2011). The tail's residence time on the filament was recorded every 30 minutes for four hours, as observed for the hot plate test. To prevent tissue damage from occurring, animals that did not demonstrate sensitivity were removed from the equipment after 30 seconds.

[0117] When we performed the tail removal test, we can observe that the control groups maintained the expected standard for the test when we compared the saline group with morphine and enkephalin, and the saline group with morphine+naloxone and enkephalin+naloxone (Figures 8 and 9).

[0118] We detected the maintenance of the analgesic effect when we compared the groups that received the peptides with the water group, in addition to maintaining similarity with the effect of morphine (Figures 10, 11 and 12). [0119] When evaluating each peptide separately, we can observe that SEQ ID NO:1 maintained statistically significant lower values in relation to mofin only at times 30 and 60, however, there was no difference when compared to the SEQ ID NO:1 +naloxone group , however, the SEQ ID NO:1 +naloxone group showed similar results to the water group (Figure 10). In figure 11 we can see that morphine only showed different results from the SEQ ID NO:2 peptide at time 30 and 60. And the SEQ ID NO:2 group when compared with SEQ ID NO:2+naloxone only showed statistically significant results at time 30, but when we compared the SEQ ID NO:2+naloxone group with saline, we observed that they presented similar results. In Figure 12 we see that morphine only presented a statistically significant result in relation to the peptide of SEQ ID NO:3 at time 30 and SEQ ID NO:3 in relation to SEQ ID NO:3 + naloxone at time 60, but the SEQ group ID NO:3 +naloxone and saline maintained rates very close to the point that there was no significant difference between the groups.

[0120] To assess whether the peptides would cause any changes in the behavioral pattern of the mice, we carried out tests to assess anxiety, coordination and balance. For this we used the open field, elevated plus maze and rotarod tests. These were carried out 30 minutes after the administration of treatments.

[0121] Open field test (TCA)

[0122] To evaluate the stimulant, depressant or anxiolytic activity of treatments, we use the TCA (BROADHURST, 1960). The equipment for carrying out this test consists of a white circular arena, 30 cm in diameter and 30 cm high. A circle divides the bottom of this arena into two parts, peripheral and central quadrants, which, in turn, are subdivided by line segments into 12 approximately equal areas.

[0123] So that the smell of urine and feces did not interfere with the test, after removing each animal, the arena was cleaned with 20% alcohol. And after 30 minutes after treatment administration, each animal was placed in the center of the equipment and observed for 5 minutes. In this test we observed the numbers of: crossings (number of quadrants covered), self-cleaning, raising the animal on its hind legs, fecal cakes and the time in seconds of total absence of movements.

[0124] TCA is a model that evaluates motor and exploratory activity, as changes in these parameters can be an indication of anxiety in animals, where CNS-stimulating substances tend to increase the behavioral parameters recorded in the model, and depressant substances tend to decrease them. Although there is conflict caused by novelty, it is natural for the animal to explore a new environment (MONTGOMERY, 1958). In this test, rodents prefer the periphery of the device, avoiding constant activities in the central part of the field. This greater activity in the central quadrants indicates less anxiety. This characteristic of remaining on the periphery, close to the walls of the equipment, is related to thigmotatism (PRUT; BELZUNG, 2003).

[0125] During the experiment, no changes were recorded in NBF, NCC and AL. Our results also demonstrated that the peptides did not change the behavior of the mice, because although the number of surveys of the SEQ ID NO:2 peptide was higher than the water and morphine group, other patterns such as an increase in the amount of NCP and NCC were not detected. , where the animals that received the peptides did not differ statistically from the saline group (Figure 13).

[0126] Elevated plus maze test (TLCE)

[0127] The TLCE (HANDLEY; MITHANI, 1984), is a wooden device in the shape of a cross, elevated 50 cm from the ground and consisting of two open arms (28.5 cm x 7.0 cm), arranged perpendicularly to two other closed arms of the same size, but surrounded by 14 cm high walls.

[0128] The mice were placed in the center of the equipment, with the face facing one of the closed arms. From that moment on, we quantified, through direct observation, for 5 minutes, the number of entries and the time spent in the open and closed arms and in the center of the equipment. Between exposures of each animal, the device was cleaned with a 20% alcohol solution.

[0129] When evaluating our results in TLCE (Figures 14 and 15), we can observe that our control who received morphine, which is a drug that when administered acutely has demonstrated anxiolytic-like actions in TLCE (MILADI-GORJI; RASHIDY- POUR; FATHOLLAHI, 2012), did not present a greater number of entries into the open arms compared to the saline group. This characteristic may be related to the group's reduced movement through the device, which can be observed in the reduced number of EBF in the morphine group. However, we must highlight that although opioids such as morphine are treated as behavioral stimulants in rodents, it has already been proven that depending on the dose and site of administration, this group of drugs can have an inhibitory and excitatory effect on motor activity (PATTI et al. , 2006). In his results evaluating the effects of pairing morphine with open arms, Oliveira (2012) also did not significantly observe classic indicators of anxiety.

[0130] When evaluating the amount of EBF, we observed the highest locomotion index presented by the groups SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and enkephalin, which ends up being reflected in the lower TBF of SEQ ID NO :1 and SEQ ID NO:2 (Figures 16,17 and 18). The assessment of entries into closed arms has been used in research as an index of locomotion, where the administration of opioids tends to cause a higher rate of entry into closed arms, in relation to the negative control group (LIPTÁK et al., 2012; REZAYOF ; ASSADPOUR; ALIJANPOUR, 2013). Furthermore, the pattern of exploration in LCE is the predominance of staying in closed arms to the detriment of open ones (CASARRUBEA et al., 2013; HANDLEY; MITHANI, 1984).

[0131] When we compare the groups that received the peptides with the saline group, we found that there was no significant difference in terms of EBA and TBA, which is positive, as it demonstrates that the administration of the peptides did not cause an increase in anxiety levels in the animals in relation to the control group (RANDALL-THOMPSON; PESCATORE; UNTERWALD, 2010; SEROVA et al„ 2013).

[0132] Rotating bar test - Rotarod

[0133] The rotating bar test (rotarod) is a test to evaluate the motor coordination and balance of animals subjected to different treatments (DUNHAM; MIYA, 1957). This equipment consists of a 3 cm wide bar, suspended 20 cm from the surface of the device and has a speed regulated in revolutions per minute (r.p.m.).

[0134] Twenty-four hours before administering treatments, all animals were conditioned to walk on the rotating bar, until they were fully familiar with the equipment. Thirty minutes after administering the treatments, each animal was placed on the bar in three cycles of two minutes each and the result was obtained by the average time in seconds each group spent in the device.

[0135] Regarding coordination and balance, no group showed changes when subjected to the rotarod test. All groups spent an average of approximately 120 s on the rotating bar, which demonstrates that all groups maintained coordination and balance.

[0136] Those skilled in the art will value the knowledge presented here and will be able to reproduce the invention in the modalities presented and in other variants and alternatives, covered by the scope of the following claims.

Claims

Claims

1. Use of a peptide characterized by being to prepare an opioid and/or antinociceptive medicine, in which the peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or combinations thereof.

2. Use of peptide, according to claim 1, characterized in that it is in the preparation of medicines for the treatment of at least one disease associated with pain selected from the group consisting of cancers; bacterial, viral, fungal and parasitic infections; autoimmune and/or inflammatory diseases; burns; and traumas.

3. Use of peptide characterized by being to prepare a medicine for the treatment of pain, cough, diarrhea, shortness of breath and hyperalgesia, wherein the peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or combinations thereof.

4. Use of peptide according to any one of the preceding claims, characterized in that it is for preparing a medicament, in which the peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or combinations thereof; wherein the dose of the peptide is 20 pM to 30 pM, preferably 26 pM.

5. Use of peptide according to any one of the preceding claims, characterized in that it is for preparing a medicament applied intravenously, intrathecally or orally, preferably intravenously or intrathecally, even more preferably intravenously.

6. Use of peptide, according to any of the previous claims, characterized in that it is to prepare a medicine for the treatment and/or control of dependence and/or tolerance to opium and/or opioids.

7. Formulation characterized by comprising at least one peptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and at least one excipient.

8. Formulation according to claim 7, characterized in that the peptide is in a concentration of 1 pM to 50 pM.

9. Formulation according to claim 7 or 8, characterized in that it is in the form of a solution, suspension or emulsion; wherein preferably the formulation is sterile.

10. Formulation according to any one of claims 7 to 9, characterized by additionally comprising an active compound selected from the group consisting of anticonvulsants, antidepressants, antipsychotics, anxiolytics, local anesthetics, corticosteroids, ketamines, non-steroidal anti-inflammatory drugs, muscle relaxant and antihypertensives.