Peptide derived from crotalphine, composition comprising the said peptide, its use and method to treat acute or chronic pain

Abstract

The present invention refers to a peptide derived from crotalphine as established by SEQ ID NO: 1, pharmaceutical composition comprising said peptide derived from crotalphine, its use and methods to treat acute or chronic pain.

Description

PEPTIDE DERIVED FROM CROTALPHINE, COMPOSITION COMPRISING THE SAID PEPTIDE, ITS USE AND METHOD TO TREAT ACUTE OR CHRONIC PAIN FIELD OF INVENTION

[0001] The present invention falls within the area of medical science. More specifically, the present invention refers to a peptide derived from crotalphine as established by SEQ ID NO: 1, pharmaceutical composition comprising said peptide derived from crotalphine, its use and methods to treat acute or chronic pain. BACKGROUND OF THE INVENTION

[0002] Pain is a global public health problem. Estimates suggest that 20% of the adult population suffers from some type of pain and 10% are newly diagnosed with chronic pain each year. It affects all populations, regardless of age, sex, income, race / ethnicity or geography (Henschke et al., 2015), interfering with various aspects of the patient's life, including sleep, recreation, family environment, occupational activities, professional activities, and some patients also present severe symptoms of depression.

[0003] Further experimental studies become relevant to expand knowledge of the molecular and cellular mechanisms involved in nociceptive processes, as well as for the development of new analgesic drugs, with greater efficacy and lower intensity of adverse effects.

[0004] The control of pain, especially chronic pain, is not yet fully effective and many clinically available compounds induce severe adverse effects. Therefore, it is considered that the search for new analgesic compounds is still necessary. In this sense, animal venoms and componentshave been explored as a source of molecules that can be studied in different disorders.

[0005] Pain, especially that resulting from chronic conditions, represents a growing public health problem as it often becomes debilitating, and resistant to therapies available on the market. These characteristics show the importance of the development of new therapies, including pharmacological therapies.

[0006] Crotalphine (CRO) is a peptide initially characterized and isolated from the venom of Crotalus durissus terrificus snakes that has shown promising activities. This peptide has a potent and long-lasting analgesic action, mediated by the activation of peripheral cannabinoid and opioid receptors.

[0007] The US Patent No. US 5,232,911, granted on August 3, 1993, in the name of VENTECH RESEARCH INC., entitled: “MIXTURE OF A NON-COVALENT HETERODIMER COMPLEX AND A BASIC AMPHIPHATIC PEPTIDE AS CYTOTOXIC AGENT” refers to a cytotoxic agent useful against malignant tumors. It provides a stable composition of matter based on the cytotoxic activity of two synergistically acting toxins whose sequence is described in the document. The basic amphipathic peptide binds to the cell membrane causing disruption of the lipid bilayer. The noncovalent heterodimer complex dissociates and the phospholyphase A2 (B) subunit binds to the cell membrane. The A subunit acts as a chaperone, preventing the nonspecific binding of phospholipase A2, therefore, it does not have enzymatic activity. Basic amphipathic peptide enhances the effect of the phospholipase A2 subunit. Cell death is caused by enzymatic hydrolysis of cell membrane phospholipids. Both the noncovalent heterodimer complex and the basicamphipathic peptide used were purified from the venoms of Crotalus durissus terrificus and Naja naja atra, respectively.

[0008] The US Patent No. US 5,232,911 discloses the sequence of 14 amino acids of the γ chain of the A subunit of the Crotoxin complex extracted from the Crotalus durissus terrificus snake venom (sequence in columns 1 to 4; SEQ ID NO: 4 and aspect 2 of US Patent No. US 5,232,911).

[0009] The Brazilian patent no. PI 0502399-8, published on May 2, 2006, and granted on May 2, 2018, in the name of FUNDAÇÃO DE AMPARO à PESQUISA DO ESTADO DE SÃO PAULO - FAPESP; BIOSINTÉTICA FARMACÊUTICA LTDA and YARA CURY, and entitled: “COMPOUNDS ANALOGOUS TO ANALGESIC PEPTIDES DERIVED FROM CROTALUS DURISSUS TERRIFICUS SNAKE POISON, THEIR USES, COMPOSITIONS, PREPARATION AND PURUFIFICATION METHODS” comprises compounds analogous to peptides with amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, including analgesic peptides derived from snakes of the Crotalus durissus terrificus species; their uses in the treatment, diagnosis and prevention of painful processes or processes mediated by opioid receptors, their compositions and their preparation and purification methods, as well as their uses in identifying analgesic compounds.

[0010] In a similar way to the US Patent No. US 5,232,911, the Brazilian patent no. PI 0502399-8 discloses the sequence of 14 amino acids from the snake venom Crotalus durissus terrificus).

[0011] The Brazilian Patent Application No. BR 10 2016 023153 1, published on May 2, 2018, in the name of FUNDAÇÃO BUTANTAN and entitled: “COMPOSITION, USE, AND METHOD FOR TREATING NEURODEGENERATIVE DISEASES” refers to acomposition comprising SEQ ID NO: 1 or 2 and one or more pharmaceutically acceptable carriers or diluents. The Brazilian Patent Application No. BR 102016 023153 1 further relates to the use of the composition or SEQ ID NO: 1 or 2 for the production of a medicament for the treatment of neurodegenerative diseases and to a method for treating neurodegenerative diseases. The Brazilian Patent Application No. BR 10 2016 023153 1 comprises compounds analogous to peptides with amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2 including analgesic peptides derived from Crotalus durissus terrificus snakes. The Brazilian Patent Application No. BR 10 2016 023153 1 demonstrates the efficacy of crotalphine in an animal model for multiple sclerosis, demonstrating that crotalphine is capable of partially reversing the motor impairment observed in this disease.

[0012] In a similar way to the US Patent No. US 5,232,911, and to the Brazilian patent no. PI 0502399-8, the Brazilian Patent Application No. BR 10 2016 023153 1 also discloses the sequence of 14 amino acids from the snake venom Crotalus durissus terrificus).

[0013] However, the present invention refers to a peptide sequence of 5 amino acids, while the aforementioned patent refers to a peptide sequence of 14 amino acids of Crotalphine. Furthermore, the present invention refers to a peptide having the sequence ZFSPEN*, where Z is a pyroglutamate and the * corresponds to an amidation, that is, the last Asn residue is a CO-NH2 and not COOH as per in the crotalphine of said patent application.

[0014] The Paper written by Aird S. D. et al, Biochem., vol.24 (25): 7054-7058 – published in 1985 and entitled “RATTLESNAKE PRESYNAPTIC NEUROTOXINS: PRIMARYSTRUCTURE AND EVOLUTIONARY ORIGIN OF THE ACIDIC SUBUNIT1” describes crotoxin and homologous crotalide presynaptic neurotoxins that consist of a toxic basic subunit and a slightly smaller, non-toxic acidic subunit. The latter, in turn, is formed by three chains, interconnected by disulfide bridges. The complete sequences of two of the three acidic subunit chains of crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, have been determined. Said article emphasizes the crotoxin B subunit and reveals that its sequence has 35 amino acids. In said article, nine oligopeptide fragments were found resulting from the cleavage of the Crotoxin B subunit sequence, none of them with analgesic effects.

[0015] However, the present invention induces a long-lasting analgesic effect in both acute inflammatory pain models and chronic pain models and comprises a peptide sequence of only 5 amino acids.

[0016] Therefore, the aforementioned article was cited only to illustrate the state of the art of the present invention.

[0017] The article written by Faure G. et al, Biochem., vol 30 (32): 8074-8083 – published in 1991 and entitled: “MULTIPLICITY OF ACIDIC SUBUNIT ISOFORMS OF CROTOXIN, THE PHOSPHOLIPASE A2 NEUROTOXIN FROM CROTALUS DURISSUS TERRIFLCUS VENOM, RESULTS FROM POSTTRANSLATIONAL MODIFICATIONS” describes different isoforms of the γ chain of the A subunit of the Crotoxin complex extracted from the venom of the Crotalus durissus terrificus snake.

[0018] The present invention differs from the article written by Faure G. et al because, although it reveals crotalphine sequences, it does not reveal that thesequence is ZFSPEN*, where Z is a pyroglutamate and the * corresponds to an amidation, that is, the last Asn residue is a CO-NH2 and not COOH as in crotalphine. Furthermore, at no point its use as an analgesic is described, much less suggested.

[0019] Therefore, the aforementioned article was cited only to illustrate the state of the art of the present invention.

[0020] The two modifications mentioned in the present invention - the introduction of Z (pyroglutamate) and amidation in the last Asn residue increase the stability of the peptide, protecting it from in vivo degradation by proteases, which reflects in the longer duration of action observed by mini-Cro of the present invention, and in the fact that mini-Cro of the present invention is stable at acidic and neutral pHs, while crotalphine is only stable at neutral pHs, but not acidic.

[0021] Although several studies have been identified on the use of compounds derived from snake venom and on the use of peptides that act on opioid receptors, such studies, however, did not elucidate the nature of the active analgesic principle present in snake venom of the Crotalus durissus terrificus species, nor its effectiveness, when administered in purified form, even orally. Such studies also did not elucidate the effectiveness of compounds analogous to this active ingredient, nor their specific action on opioid receptors. SUMMARY OF THE INVENTION

[0022] The present invention contemplates a composition comprising a crotalphine peptide derivative presenting two modifications to the crotalphine structurerevealed by prior art, that is, the introduction of Z (pyroglutamate) and amidation in the last Asn residue, thus increasing the stability of the peptide, protecting it from in vivo degradation by proteases, which reflects the longer duration of action observed by the compound of the present invention, and the fact that the compound of the present invention is stable at acidic and neutral pHs, while the Crotalphine from prior art is only stable at neutral pHs, but not at acidic pHs.

[0023] In a first aspect, the present invention relates to a composition comprising SEQ ID NO: 1 and one or more pharmaceutically acceptable carriers or diluents.

[0024] In another aspect, the present invention relates to the use of the composition for the production of a medicament for the treatment of acute or chronic pain. BRIEF DESCRIPTION OF FIGURES

[0025] The object of the invention, together with additional advantages thereof, can be better understood by referring to the attached figures and the following descriptions:

[0026] Figure 1 shows the amino acid sequence of crotalphine of the prior art;

[0027] Figure 2 shows a dose-response curve of exemplary compositions of the present invention (small-CRO) in an acute inflammatory pain model. The animals underwent electronic behavioral evaluation by von Frey, before and after intraplantar injection of carrageenan treated with saline or mini-CRO orally. The results are expressed as the mean (± SEM). n= 5. * p < 0.05 indicates statistical difference between initial and final measurements. # p < 0.05 indicates statistical difference in relation to the Cg+ Saline p.o. group. The two-way ANOVA test was used followed by the Sidak test;

[0028] Figure 3 shows the animals that underwent electronic behavioral assessment by von Frey, before (at different times) and after the intraplantar injection of carrageenan, treated with saline, CRO or mini-CRO orally. The results are expressed as the mean (± SEM). n= 5. * p < 0.05 indicates statistical difference between initial and final measurements. # p < 0.05 indicates statistical difference in relation to the Cg + Saline p.o. group. The two-way ANOVA test was used followed by the Sidak test;

[0029] Figure 4 shows the animals that underwent behavioral evaluation using the Hargreaves plantar test, before and after the intraplantar injection of carrageenan, treated with saline or mini-CRO orally. The results are expressed as the mean (± SEM). n= 5-6. * p < 0.05 indicates statistical difference between initial and final measurements. # p < 0.05 indicates statistical difference in relation to the Cg + Saline p.o. group. The two-way ANOVA test was used followed by the Sidak test;

[0030] Figure 5 shows the animals that were treated on the 14th day after CCI surgery (indicated by the arrow) orally and subjected to electronic behavioral assessment by von Frey 1 and every 24 hours after administration. The results are expressed as the mean (± SEM). n= 5. * p < 0.05 indicates statistical difference in relation to the Sham + Saline p.o. group. # p < 0.05 indicates statistical difference in relation to the CCI group + Salina p.o. The two-way ANOVA test was used followed by the Tukey test;

[0031] Figure 6 shows animals that were treated with saline or mini-CRO orally and evaluated 1 and 3 hours afteradministration. The results are expressed as the mean (± SEM). n=6. No statistical difference was identified between the groups. The two-way ANOVA test was used followed by the Sidak test;

[0032] Figure 7 shows reversed-phase chromatography on a C18 column on the HPLC system (Shimadzu) of mini-Cro submitted to different temperatures. Gradient from 10 - 60% B in 20 min. Flow 1 mL / min. Absorbance at 214 nm. Restek Ultra C-185µm column, 150 × 4.6 mm;

[0033] Figure 8 shows reversed-phase chromatography on a C18 column on the HPLC system (Shimadzuo) of mini-Cro submitted to different temperatures. Gradient from 10 - 60% B in 20 min. Flow 1 mL / min. Absorbance at 214 nm. Restek Ultra C-185µm column, 150×4.6mm

[0034] Figure 9 shows the animals that underwent electronic behavioral assessment by von Frey, before and after the intraplantar injection of carrageenan, treated with saline, CRO or mini-CRO orally. The results are expressed as the mean (± SEM). n= 5. * p < 0.05 indicates statistical difference between initial and final measurements. # p < 0.05 indicates statistical difference in relation to the Cg + Saline p.o. group. The two-way ANOVA test was used followed by the Sidak test; and

[0035] Figure 10 shows reversed phase chromatography on a C18 column on the HPLC system (Shimadzu) of mini-Cro submitted to different pHs. Gradient from 10 - 60% B in 20 min. Flow 1 mL / min. Absorbance at 214 nm. Restek Ultra C-18 5µm column, 150 × 4.6 mm. DETAILED DESCRIPTION OF THE INVENTION

[0036] Although the present invention may be susceptible to different embodiments, there is shown in thedrawings and in the following detailed discussion a preferred embodiment with the understanding that the present description should be considered an exemplification of the principles of the invention and is not intended to limit the present invention to what has been illustrated and described here. Peptides

[0037] By their nature, the venoms of predatory animals are generally potent and fast-acting and require a degree of stability of their constituent peptides, and this evolutionary requirement has selected particular classes of peptides that make them excellent for drug design and development (Craik et al., 2013).

[0038] Peptides are generally small molecules, formed by short chains of amino acids (50 amino acids or less), capable of inducing many of the functions of proteins. They generally have advantages when compared to proteins, which include ease of production in -mammalian cells or bacteria or by solid-phase peptide synthesis, low cost of synthesis, and they are stable in a wide variety of conditions, which allows for long-term storage and easy handling. Furthermore, they are considered good strategies for treating various diseases because they are generally small, potent, specific molecules and do not generate toxic metabolites (Pérez de Vega et al., 2018). Some disadvantages may be related to a short half-life in plasma and susceptibility to degradation by proteases.

[0039] Given this, peptides have become progressively important for the development of pharmaceutical products against a variety of diseases, particularly due to technological advances related tomolecule design and production processes, which currently allow the development of peptides with specific physicochemical properties necessary for appropriate formulations with determined “bioperformance” characteristics (D'Addio et al., 2016). In this sense, structural modifications of proteins / peptides can influence solubility, stability, lipophilicity, enzymatic susceptibility and affinity for the target, consequently interfering with its therapeutic potency (Bolhassani, 2019).

[0040] Furthermore, changes in physicochemical properties influence the absorption of the compound as well as the ability to cross membranes, which can either improve binding or time of action on the specific target or reduce adverse effects of molecules, reducing, for example, the ability to cross the blood-brain barrier membrane (Komin et al., 2017). Crotalphine sequence is: - < E F S P E N C Q G E S Q P C (with a disulfide bridge connecting cysteines at positions 7 and 14) – SEQ ID NO: 2 Mini-crotalphine (mini-Cro) of the present invention

[0041] Mini-cro of the present application has the peptide sequence defined as ZFSPEN* (SEQ ID NO: 1), where Z is a pyroglutamate and the * corresponds to an amidation, that is, the last Asn residue is a CO-NH2 and not COOH (as in the original crotalphine) at the C-terminus.

[0042] The two mentioned modifications - the introduction of Z (pyroglutamate) and amidation in the last Asn residue - increase the stability of the peptide, protecting it from in vivo degradation by proteases which reflects in the longer duration of action.Crotalphine x derivative (Mini-CRO) of the present invention

[0043] Crotalphine (CRO), a peptide of 14 amino acids, containing a disulfide bridge and a pyroglutamic acid (<E F S P E N C Q G E S Q P C>) shown in the figure 1, measuring approximately 1.5 kDa (figure 1). It is an analgesic compound first identified in the venom of the snake Crotalus durissus terrificus (Konno et al., 2008).

[0044] Crotalphine induces long-lasting analgesia (up to 5 days) in an acute pain model (Konno et al., 2008) and in a neuropathic pain model induced by chronic constriction of the sciatic nerve lasting 2 to 3 days (Gutierrez et al., 2012), with no adverse effects identified to date. Although CRO-induced analgesia is reversed by opioid antagonists, this peptide is not similar to any other opioid described in the literature.

[0045] Based on its sequence, 8 analogues that correspond to both a portion of the leader molecule (CRO), as well as compounds that underwent specific modifications were synthesized. From this, the inventors of the present application arrived at a peptide here called mini-CRO, whose sequence is ZFSPEN*, where Z is a pyroglutamate and the * corresponds to an amidation, that is, the last Asn residue is a CO-NH2 and not COOH as in the original crotalphine) at the C-terminus. In vivo assays Example 1 - Dose-response curve of mini-crotalphine in an acute inflammatory pain model

[0046] The doses used in these trials were based on the effective doses of crotalphine, having been calculated at the same molar concentration.

[0047] The animals were subjected to the electronicvon Frey test in the presence of acute inflammation induced by carrageenan and 1 hour before the final measurement they were orally treated with mini-CRO at concentrations of 0.064, 0.32 and 1.62 μM (which correspond to 0.45, 2.28 and 11.44 µg / kg).

[0048] The results demonstrated that all doses administered induced a pronounced analgesic effect, with doses of 0.064 and 0.32 µM partially reversing it, and the highest dose used was able to completely reverse the pain induced by carrageenan, since there is no difference between the final measurement and the initial measurement for this group (Figure 2). Example 2 - Evaluation of the duration of effect of mini- crotalphine of the present invention in an acute inflammatory pain model

[0049] Considering the results obtained in the previous experiment, mini-CRO was used at a concentration of 1.62 µM, a dose that completely reversed the pain induced by the model.

[0050] It is known that CRO in a single dose is capable of causing analgesia for up to 5 days (Konno et al., 2008). In this sense, to evaluate the duration of the effect of mini-CRO in this model, the animals were evaluated 1, 72, 96, 120, 144 and 168 hours after treatments. Carrageenan was intraplantarly administered 3 hours before each final measurement.

[0051] It is important to highlight that different groups of rats were used, as each measurement was performed at the peak of pain. For this, carrageenan was intraplantarly administered 3 hours before each final measurement. As shown in figure 3, mini-CRO completely reverses carrageenan-induced pain 1, 72 and 96 h after its administration (Figure 3 A, B, C), with partial analgesia observed 120 and 144 hours after treatment (Figures 3 D and E). This effect was not observed 168 h (7 days) after treatment (Figure 3 F). Example 3 - Evaluation of the effect of mini-crotalphine on thermal hyperalgesia

[0052] After determining the dose to be used in a model of acute inflammatory pain, the inventors of the present invention investigated the effect of mini-CRO on thermal hyperalgesia.

[0053] For this, an initial measurement was obtained, then carrageenan was administered intraplantarly, 2 hours later the animals were treated with saline or mini- CRO and 1 hour after treatment (and 3 hours after carrageenan injection) the animals were evaluated again, and the final measurement was determined.

[0054] The data obtained demonstrate that mini-CRO induces partial reversion of carrageenan-induced pain (Figure 4). Example 4 - Dose-response curve of mini-crotalphine of the present invention in a neuropathy model.

[0055] To evaluate the analgesic effect of mini- crotalphine as well as its duration, the neuropathy model was induced by chronic constriction of the sciatic nerve. On day 0, the animals underwent behavioral testing, and afterward underwent chronic sciatic nerve constriction surgery (CCI). The animals were evaluated for 17 days, and on the 14th day after surgery (the period in which neuropathy is already present) were treated with different concentrations of the compound (1.62, 3.25, and 6.51 μM) or saline. As controls, sham-operated animals (sham) and naïveanimals (animals that had not undergone any type of surgical manipulation) were used.

[0056] The data demonstrate that the nociceptive threshold of sham-operated animals remained the same, without pain, until the end of the experiment. Differently, operated animals that received only saline showed a reduction in this threshold throughout the entire period evaluated.

[0057] Animals treated with a concentration of 3.25 μM of mini-CRO of the present invention showed complete reversal of the painful condition, which lasted for more than 24 hours. After 48 hours, the analgesia induced by the compound is still seen, but partially, and 72 hours after treatment this effect is no longer observed. In contrast, concentrations of 1.62 and 6.51 μM of the peptide were not able to reverse the pain induced by the model (figure 5).

[0058] Furthermore, the data obtained demonstrated that the peptide does not alter the nociceptive threshold of sham animals, confirming that, regardless of the model used, its effect is only observed in the face of sensitization. Example 5 - Evaluation of the effect of mini-crotalphine in naïve animals in the Open Field (OF) test

[0059] The OF test is a common measure of exploratory behavior and locomotor in rats and mice, used to evaluate the general activity of these animals and to investigate whether compounds would interfere with the spontaneous activity of animals.

[0060] The animals were treated orally with a concentration of 3.25 μM miniCRO of the present invention, the highest concentration capable of completely reversing the animals' pain and evaluated 1 and 3 hours after administration.

[0061] The results show that the compound does not interfere with the animals' locomotion and exploration capabilities in both periods evaluated (figure 6). In vitro assays Example 6- Forced degradation test of the Mini-CRO of the present invention: Temperature

[0062] According to the ANVISA RDC No. 53, the study of forced degradation allows the possible generation of degradation products through exposure of the compound to stress conditions, such as temperature, acid / base hydrolysis, among others. In this way, it is possible to develop stability-indicative methods with adequate specificity and selectivity, as well as provide information about the possible degradation routes of a given product.

[0063] To evaluate its stability, the mini-CRO of the present invention was exposed to different temperatures, these being freezing at -20oC, ambient temperature at 22oC - 25oC, human body temperature at 37oC and a higher temperature at 42oC - 45oC. The results obtained demonstrate that the peptide is stable, that is, it remains intact under the different conditions tested (figure 7). Example 7 - Forced degradation test of the mini-CRO of the present invention: pH

[0064] Considering that the activity and stability of several molecules depend on the pH at which they are found, our next step was to investigate the stability of the mini-CRO of the present invention at different pHs. To this end, the peptide was incubated in a water bath at 37ºC for 24 hours in buffers containing different pHs, and then the samples were evaluated by HPLC.

[0065] The results demonstrate that mini-CRO of thepresent invention is stable in acidic pH, however, in more alkaline conditions, from pH 9 onwards, it does not maintain its stability (figure 8). Comparison of the effect of mini-Cro with Crotalphine in terms of duration of effect and stability Example 8 - Duration of the analgesic effect

[0066] With the aim of comparing the duration of the analgesic effect of mini-Cro of the present invention with crotalphine, rats were treated with saline, CRO or mini-CRO, both at the same molar concentration, and evaluated 1, 72, 96, 120, 144 and 168 hours after treatments. Carrageenan was intraplantarly administered 3 hours before each final measurement.

[0067] The obtained data show that after a single administration of mini-CRO, the analgesic effect was observed for up to 144 hours (6 days) after administration and was no longer observed on the 7th day after treatment. The analgesic effect of crotalphine was observed for up to 72 hours (Figure 9). Example 9 - Stability at different pHs

[0068] As previously mentioned, the stability of several molecules depends on the pH at which they are located. Therefore, the inventors of the present application investigated the stability of mini-CRO and Crotalphine at different pHs.

[0069] To this end, the peptides were incubated in a water bath at 37ºC for 24 hours in buffers containing different pHs, and then the samples were evaluated by HPLC.

[0070] The results demonstrate that while Crotalphine maintains its stability only at pHs 7.4 - 8, being degraded in all other conditions (figure 10) mini-CROis stable at acidic pH, losing stability only in more alkaline conditions, starting at pH 9, (Figure 8).

[0071] In a first aspect, the present application relates to a peptide sequence derived from crotalphine wherein it is as established by SEQ ID NO: 1, where SEQ ID NO: 1 is ZFSPEN*, where Z is a pyroglutamate and the * corresponds to an amidation, that is, the last Asn residue is a CO-NH2.

[0072] The introduction of Z (pyroglutamate) and amidation at the last Asn residue at the SEQ ID NO: 1 (Mini- CRO) increase the stability of the peptide, protecting it from degradation in vivo by proteases, which reflected in the longer duration of action.

[0073] Said peptide sequence of the present invention (SEQ ID NO: 1) is stable at acidic and neutral pHs. Pharmaceutical composition

[0074] In a second aspect of the present application there is disclosed a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers or diluents and the peptide sequence derived from crotalphine as defined in aspect 1 (SEQ ID NO: 1), its salts or solvates.

[0075] The pharmaceutical composition of the present application may be in the form of solution, suspension, paste, gel, capsule, tablet, lozenge, tablet, powder, granule, lyophile, controlled release systems, microparticles, micro or nanospheres, lysosomes or associated with organic coating.

[0076] Examples of pharmaceutical forms, carriers, diluents and routes of administration comprised by the present invention are described (but not limited) in thebook Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pennsylvania, USA.

[0077] Carriers or diluents, as used in the present invention, refer to a non-toxic, inert solid, semisolid liquid excipient, diluent, auxiliary formulation of any type, such as saline and water. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches, such as corn starch and potato starch, cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, cyclodextrin; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean seed oil; glycols such as propylene glycol; polyols such as glycerin glycol, sorbitol, mannitol and polyethylene; esters, such as ethyl laurate, ethyl oleate, agar; buffering agents such as aluminum hydroxide and magnesium hydroxide; alginic acid; isotonic saline, Ringer's solution; buffer solutions of ethyl alcohol and phosphate, oily emulsion in water containing mycobacteria killed by heat or components of their cell wall (complete Freund's adjuvant), as well as other compatible non-toxic substances used in pharmaceutical formulations.

[0078] The pharmaceutical composition of the present application may be for oral, intramuscular, intravenous, subcutaneous, topical, pulmonary, intranasal, buccal, rectal, sublingual, intraperitoneal, intradermal or intrathecal use. Use

[0079] The third aspect of the present application relates to the peptide sequence as defined in aspect 1 or pharmaceutical composition as defined in aspect 2, for thepreparation of a medicament for the treatment of acute or chronic pain.

[0080] The use can be as an analgesic pharmaceutical medicine, preferably as an oral analgesic pharmaceutical medicine, even more preferably as a long-lasting analgesic pharmaceutical medicine of up to 5 days. Chronic or acute pain, in the terms of the above mentioned paragraph, is selected from the group comprising cancer pain, neuropathic pain such as trigeminal neuralgia, sympathetic dystrophy, post-herpetic neuralgia, phantom limb pain, post-herpetic pain, stroke, diabetic neuropathy, pain associated with neoplasms, fibromyalgia, dental pain, dysmenorrhea, renal, menstrual or biliary colic, joint pain, arthritis including rheumatoid arthritis or degenerative arthritis, intraocular hypertension, post-arthroscopy pain, pain after gynecological laparoscopy, pain produced by percutaneous nephrolithotomy, pain after radical retropubic prostectomy, post-thoracotomy pain, post-tonsillectomy pain in pediatric patients, post-hysterectomy pain, pain after cesarean section or burns, cocaine or opioid dependence, cell proliferation, small cell lung carcinoma, depression and psychosis, inflammation, conditions associated with enlargement of angiogenesis, wounds, coronary ischemic diseases, neurodegenerative diseases, Parkinson's disease and dyskinesias, hepatic encephalopathy, cognitive diseases, Alzheimer's, pruritus due to hepatic cholestasis or hyperinsulinemia in women with polycystic ovary. Neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Autoimmune Diseases, hepatitis and inflammatory and degenerativerheumatisms, including rheumatoid arthritis. METHOD TO TREAT ACUTE OR CHRONIC PAIN

[0081] The fourth aspect of the present application relates to a method for treating chronic or acute pain, administering the peptide as defined in aspect 1 or the composition as defined in aspect 2 to a patient suffering from said disease.

[0082] Chronic or acute pain, in the terms of the above mentioned paragraph, is selected from the group comprising cancer pain, neuropathic pain such as trigeminal neuralgia, sympathetic dystrophy, post-herpetic neuralgia, phantom limb pain, post-herpetic pain, stroke, diabetic neuropathy, pain associated with neoplasms, fibromyalgia, dental pain, dysmenorrhea, renal, menstrual or biliary colic, joint pain, arthritis including rheumatoid arthritis or degenerative arthritis, intraocular hypertension, post- arthroscopy pain, pain after gynecological laparoscopy, pain produced by percutaneous nephrolithotomy, pain after radical retropubic prostectomy, post-thoracotomy pain, post- tonsillectomy pain in pediatric patients, post-hysterectomy pain, pain after cesarean section or burns, cocaine or opioid dependence, cell proliferation, small cell lung carcinoma, depression and psychosis, inflammation, conditions associated with enlargement of angiogenesis, wounds, coronary ischemic diseases, neurodegenerative diseases, Parkinson's disease and dyskinesias, hepatic encephalopathy, cognitive diseases, Alzheimer's, pruritus due to hepatic cholestasis or hyperinsulinemia in women with polycystic ovary. Neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis,Autoimmune Diseases, hepatitis and inflammatory and degenerative rheumatisms, including rheumatoid arthritis.

[0083] The invention therefore provides the following aspects / embodiments:

[0084] 1. Peptide sequence derived from crotalphine wherein it is as established by SEQ ID NO: 1.

[0085] 2. Peptide sequence according to aspect 1, wherein the sequence SEQ ID NO: 1 is ZFSPEN*, where Z is a pyroglutamate and the * corresponds to an amidation, that is, the last Asn residue is a CO-NH2.

[0086] 3. Peptide sequence according to aspects 1 to 2, wherein the introduction of Z (pyroglutamate) and amidation in the last Asn residue increase the stability of the peptide, protecting it from in vivo degradation by proteases, which reflects in the longer duration of action.

[0087] 4. Peptide sequence according to aspects 1 to 3, wherein the said peptide is stable at acidic and neutral pHs.

[0088] 5. Pharmaceutical composition containing one or more pharmaceutically acceptable carriers or diluents and peptide sequence derived from crotalphine as defined in aspect 1, their salts or solvates.

[0089] 6. Pharmaceutical composition according to aspect 5, being in the form of solution, suspension, paste, gel, capsule, tablet, powder, granule, lyophile, controlled release systems, microparticles, micro or nanospheres, lysosomes or associated with organic coating.

[0090] 7. Pharmaceutical composition according to aspects 5 to 6, wherein it is for oral, intramuscular, intravenous, subcutaneous, topical, pulmonary, intranasal, buccal, rectal, sublingual, intraperitoneal, intradermal orintrathecal use.

[0091] 8. Pharmaceutical composition according to aspect 7, wherein it is for oral use.

[0092] 9. Use of peptide sequence as defined in aspect 1 or pharmaceutical composition as defined in aspect 5 wherein it is for preparation of a medicament to treat acute or chronic pain.

[0093] 10. Use according to aspect 9, wherein the medicine is used as analgesic pharmaceutical medicament.

[0094] 11. Use according to aspect 10, wherein the medicine is used as oral analgesic pharmaceutical medicaments.

[0095] 12. Use according to aspects 9 to 11, wherein the medicine is used as long-lasting analgesic pharmaceutical medicaments.

[0096] 13. Use according to aspect 12, wherein the medicine is used as analgesic pharmaceutical medicaments lasting up to 5 days.

[0097] 14. Use according to aspects 9 to 13, wherein the chronic or acute pain is selected from the group comprising of cancer pain, neuropathic pain such as trigeminal neuralgia, sympathetic dystrophy, post-herpetic neuralgia, pain phantom limb pain, post-stroke, diabetic neuropathy, pain associated with neoplasms, fibromyalgia, dental pain, dysmenorrhea, renal, menstrual or biliary colic, joint pain, arthritis including rheumatoid arthritis or degenerative arthritis, intraocular hypertension, pain post-arthroscopy, pain after gynecological laparoscopy, pain produced by percutaneous nephrolithotomy, pain after radical retropubic prostectomy, pain post-thoracotomy, pain post- tonsillectomy in pediatric patients, pain post-hysterectomy,pain after cesarean section or burns, dependence on cocaine or opioids, cell proliferation, small cell lung carcinoma, depression and psychosis, inflammation, conditions associated with increased angiogenesis, wounds, coronary ischemic diseases, neurodegenerative diseases, Parkinson's disease and dyskinesias, hepatic encephalopathy, cognitive diseases, Alzheimer's, pruritus due to hepatic cholestasis, or hyperinsulinemia in women with polycystic ovary.

[0098] 15. Use according to aspect 14, wherein the neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Autoimmune diseases, hepatitis and inflammatory and degenerative rheumatisms, including rheumatoid arthritis.

[0099] 16. Method for treating chronic or acute pain, wherein it is for administering the peptide as defined in aspect 1 or the composition as defined in aspect 5 to a patient suffering from said disease.

[0100] 17. Method according to aspect 16, wherein the chronic or acute pain is selected from the group comprising of cancer pain, neuropathic pain such as trigeminal neuralgia, sympathetic dystrophy, post-herpetic neuralgia, pain phantom limb pain, post-stroke, diabetic neuropathy, pain associated with neoplasms, fibromyalgia, dental pain, dysmenorrhea, renal, menstrual or biliary colic, joint pain, arthritis including rheumatoid arthritis or degenerative arthritis, intraocular hypertension, pain post-arthroscopy, pain after gynecological laparoscopy, pain produced by percutaneous nephrolithotomy, pain after radical retropubic prostectomy, pain post-thoracotomy, pain post- tonsillectomy in pediatric patients, pain post-hysterectomy,pain after cesarean section or burns, dependence on cocaine or opioids, cell proliferation, small cell lung carcinoma, depression and psychosis, inflammation, conditions associated with increased angiogenesis, wounds, coronary ischemic diseases, neurodegenerative diseases, Parkinson's disease and dyskinesias, hepatic encephalopathy, cognitive diseases, Alzheimer's, pruritus due to hepatic cholestasis, or hyperinsulinemia in women with polycystic ovary.

[0101] 18. Method according to aspect 17, wherein the neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Autoimmune diseases, hepatitis and inflammatory and degenerative rheumatisms, including rheumatoid arthritis.

[0102] Thus, although only some embodiments and exemplifications of the present invention have been shown, it will be understood that various omissions, substitutions and changes can be made by a person skilled in the art, without departing from the scope of the present invention.

[0103] It is expressly anticipated that all combinations of elements that perform the same function in substantially the same way, to achieve the same results, are within the scope of the invention. Substitutions of elements from one described embodiment to another are also fully intended and contemplated.

[0104] It is also necessary to understand that the drawings are not necessarily to scale, but that they are only conceptual in nature. It is therefore intended to be limited, as indicated by the scope of the appended aspects.REFERENCES

[0105] Bolhassani, A. (2019). Improvements in chemical carriers of proteins and peptides. Cell Biology International, 9999, 1–16.

[0106] Craik, D. J., Fairlie, D. P., Liras, S., & Price, D. (2013). The Future of Peptide-based Drugs. Chemical Biology & Drug Design, 81(1), 136–147.

[0107] D’Addio, S. M., Bothe, J. R., Neri, C., Walsh, P. L., Zhang, J., Pierson, E., Mao, Y., Gindy, M., Leone, A., & Templeton, A. C. (2016). New and Evolving Techniques for the Characterization of Peptide Therapeutics. Journal of Pharmaceutical Sciences, 105(10), 2989–3006.

[0108] Gutierrez, Vanessa P, Zambelli, V. O., Picolo, G., Chacur, M., Sampaio, S. C., Brigatte, P., Konno, K., & Cury, Y. (2012). The peripheral L-arginine-nitric oxide-cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats. Behavioural Pharmacology, 23(1),

[0109] Henschke, N., Kamper, S. J., & Maher, C. G. (2015). The epidemiology and economic consequences of pain. Mayo Clinic Proceedings, 90(1), 139–147.

[0110] Komin, A., Russell, L. M., Hristova, K. A., & Searson, P. C. (2017). Peptide-based strategies for enhanced cell uptake, transcellular transport, and circulation: Mechanisms and challenges. Advanced Drug Delivery Reviews,110–111,

[0111] Konno, K., Picolo, G., Gutierrez, V. P., Brigatte, P., Zambelli, V. O., Camargo, A. C. M., & Cury, Y. (2008). Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus. Peptides, 29(8), 1293–1304.

[0112] Lambert, G. A., Mallos, G., & Zagami, A. S. (2009). Von Frey’s hairs - a review of their technology and use - a novel automated von Frey device for improved testing for hyperalgesia. Journal of Neuroscience Methods, 177(2), 420–426. https: / / doi.org / 10.1016 / j.jneumeth.2008.10.033

[0113] Pérez de Vega, M. J., Ferrer-Montiel, A., & González-Muñiz, R. (2018). Recent progress in non-opioid analgesic peptides. Archives of Biochemistry and Biophysics, 660(October), 36–52.

[0114] Pogatzki-Zahn, E., Segelcke, D., & Zahn, P. (2018). Mechanisms of acute and chronic pain after surgery: update from findings in experimental animal models. Current Opinion in Anaesthesiology, 31(5), 575–585..

Claims

AMENDED CLAIMS received by the International Bureau on 05 November 2025 (05.11.2025)1. Peptide sequence derived from crotalphine characterized by the fact that it's sequence is ZFSPEN*, where Z is a pyroglutamate and the * corresponds to an amidation, that is, the last Asn residue is a CO-NH2.

2. Pharmaceutical composition characterized by containing one or more pharmaceutically acceptable carriers or diluents and peptide sequence derived from crotalphine as defined in claim 1, their salts or solvates.

3. Pharmaceutical composition according to claim 2, characterized by being in the form of solution, suspension, paste, gel, capsule, tablet, powder, granule, lyophile, controlled release systems, microparticles, micro or nanospheres, lysosomes or associated with organic coating.

4. Pharmaceutical composition according to claims 2 or 3, characterized in that it is for oral, intramuscular, intravenous, subcutaneous, topical, pulmonary, intranasal, buccal, rectal, sublingual, intraperitoneal, intradermal or intrathecal use.

5. Pharmaceutical composition according to claim 4, characterized in that it is for oral use.

6. Use of peptide sequence as defined in claim 1 or pharmaceutical composition as defined in claim 2 characterized by being in the preparation of a medicament to treat acute or chronic pain.

7. Use, according to claim 6, characterized in that the medicament is as analgesic pharmaceutical medicament.

8. Use, according to claim 7, characterized in that the medicament is used as oral analgesic pharmaceutical medicaments .

9. Use, according to any of claims 6 to 8, characterizedin that the medicament is used as long-lasting analgesic pharmaceutical medicaments.

10. Use, according to claim 9, characterized in that the medicament is used as analgesic pharmaceutical medicaments lasting up to 5 days.

11. Use, according to any of claims 6 to 10, characterized in that the chronic or acute pain is selected from the group comprising of cancer pain, neuropathic pain such as trigeminal neuralgia, sympathetic dystrophy, postherpetic neuralgia, pain phantom limb pain, post-stroke, diabetic neuropathy, pain associated with neoplasms, fibromyalgia, dental pain, dysmenorrhea, renal, menstrual or biliary colic, joint pain, arthritis including rheumatoid arthritis or degenerative arthritis, intraocular hypertension, pain post-arthroscopy, pain after gynecological laparoscopy, pain produced by percutaneous nephrolithotomy, pain after radical retropubic prostectomy, pain post-thoracotomy, pain post-tonsillectomy in pediatric patients, pain post-hysterectomy, pain after cesarean section or burns, dependence on cocaine or opioids, cell proliferation, small cell lung carcinoma, depression and psychosis, inflammation, conditions associated with increased angiogenesis, wounds, coronary ischemic diseases, neurodegenerative diseases, Parkinson's disease and dyskinesias, hepatic encephalopathy, cognitive diseases, Alzheimer's, pruritus due to hepatic cholestasis, or hyperinsulinemia in women with polycystic ovary.

12. Use, according to claim 11, characterized in that neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Autoimmunediseases, hepatitis and inflammatory and degenerative rheumatisms, including rheumatoid arthritis.

13. Method for treating chronic or acute pain, characterized in that it is for administering the peptide as defined in claim 1 or the composition as defined in claim 2 to a patient suffering from said disease.

14. Method, according to claim 13, characterized in that the chronic or acute pain is selected from the group comprising of cancer pain, neuropathic pain such as trigeminal neuralgia, sympathetic dystrophy, post-herpetic neuralgia, pain phantom limb pain, post-stroke, diabetic neuropathy, pain associated with neoplasms, fibromyalgia, dental pain, dysmenorrhea, renal, menstrual or biliary colic, joint pain, arthritis including rheumatoid arthritis or degenerative arthritis, intraocular hypertension, pain post-arthroscopy, pain after gynecological laparoscopy, pain produced by percutaneous nephrolithotomy, pain after radical retropubic prostectomy, pain post-thoracotomy, pain posttonsillectomy in pediatric patients, pain post-hysterectomy, pain after cesarean section or burns, dependence on cocaine or opioids, cell proliferation, small cell lung carcinoma, depression and psychosis, inflammation, conditions associated with increased angiogenesis, wounds, coronary ischemic diseases, neurodegenerative diseases, Parkinson's disease and dyskinesias, hepatic encephalopathy, cognitive diseases, Alzheimer's, pruritus due to hepatic cholestasis, or hyperinsulinemia in women with polycystic ovary.

15. Method, according to claim 14, characterized in that neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis,Autoimmune diseases, hepatitis and inflammatory and degenerative rheumatisms, including rheumatoid arthritis.

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