MXPA01010732A

MXPA01010732A - Neuronal exocytosis inhibiting peptides and cosmetic and pharmaceutical compositions containing said peptides

Info

Publication number: MXPA01010732A
Application number: MXPA/A/2001/010732A
Authority: MX
Inventors: Mira Clara Blanes; Hernandez Mercedes Llobregat; Ballester Gregorio Joaquin Fernandez; Cases Rosa Planells; Montiel Antonio Vicente Ferrer; Bover Salvador Viniegra; Castell Teresa Carbonell; Paya Enrique Perez
Original assignee: Lipotec Sa
Priority date: 1999-04-23
Filing date: 2001-10-23
Publication date: 2002-06-05

Abstract

The peptide has a sequence of 3 to 30 adjacent aminoacids from the amino end of protein SNAP-25 and is useful as neuronal exocytosis inhibitor. The cosmetic and pharmaceutical compositions contain said peptide and optionally one or more peptides from the carboxyl end of SNAP-25. Said compositions are suitable for the treatment of facial wrinkles and asymmetry and pathological neuronal exocytosis-mediated pathological disorders and alterations.

Description

PEPTIDES INHIBITORS OF THE NEORONAL EXODTOSIS, COSMETIC AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM FIELD OF THE INVENTION This invention relates to peptides derived from the amino terminus of the SNAP-25 protein, useful as inhibitors of neuronal exocytosis, and their use in cosmetic and / or therapeutic applications, optionally together with a peptide derived from the carboxyl terminus of the SNAP-25 protein.

BACKGROUND OF THE INVENTION The base or mechanism for the formation of facial wrinkles is a tension of the muscles of the epidermis that drag the skin inwards. This muscular tension is the result of an overactivity of the nerves that innervate the physical muscles. Nervous hyperactivity is characterized by an uncontrolled and excessive release of neurotransmitters that excite muscle fibers. Therefore, the molecules that control the neuronal exocytogenes contribute to relax muscle tension and, consequently, to eliminate wrinkles. Botulinum toxins are a family of bacterial neurotoxins produced by Clostridium botulinum (1) [see the section on BIBLIOGRAPHY]. Seven different serotypes are known (serotypes A, B, C, D, E, F and G) with an average molecular weight of 150 kDa. These toxins inhibit the exocytosis of acetylcholine in the neuromuscular junction (einapsis neuron-muscle) of skeletal muscle (1). At the molecular level, botulinum toxins are proteases that degrade neuronal proteins that are involved in the mechanism of exocytosis activated by the calcium ion (1-3). For example, botulinum toxin A, the most commonly used in clinical and cosmetic [for its applications in the SUBSTITUTE SHEET (RULE 26) elimination of facial wrinkles and facial asymmetry, and to smooth the symptomatology of spasmodic diseases] truncates the SNAP-25 neuronal protein. This protein (SNAP-25) plays a key role in neurosecretion since it is involved in the formation of a protein complex (known as the SNARE or fusion complex) that directs and controls the release of acetylcholine accumulated in vesicles. The nucleus of this fusion complex is the proteins SNAP-25 and syntaxin, located in the presynaptic plasma membrane, and the synaptobrevin protein (or VAMP), located in the vesicular plasma membrane (4,5). The main function of the fusion complex is to bring the neurotransmitter-laden vesicle (acetylcholine) into contact with the presynaptic plasma membrane (4,5). In this way, in response to an elevation of the calcium concentration, the fusion of both plasma membranes will be favored, thus producing the release of the neurotransmitter. Therefore, said SNARE protein complex of berthing and vesicular fusion constitutes a key target to control neurosecretion. The truncation of any of the proteins that form the fusion complex prevents its assembly and, therefore, inhibits vesicular release and neuronal exocytosis. The potency of botulinum toxins and, in particular, serotype A (BOTOX®) inhibiting neurosecretion, as well as their neuronal selectivity (only act on neurons) is being widely exploited therapeutically to correct spasmodic ailments such as dystonias, strabismus, tics , blepharospasm, torticollis, etc. (6-13). Botulinum toxin A (batulin A) is also an effective agent in the elimination of facial wrinkles and facial asymmetry. In fact, the administration of BOTOX® is the first effective therapy that does not require surgical intervention to eliminate the signs SUBSTITUTE SHEET (RULE 26) of aging (6,7). The therapeutic and cosmetic treatment with BOTOX® consists of the localized injection of pharmaceutical preparations (botulinum complex A-hemagglutinin, 500 kDa) diluted in the areas where muscle tension is located. The paralytic effects of the toxin are reversible with a mean duration of 6 months (6, 7). The treatment, therefore, requires the repeated injection of BOTOX®. The main problem with this treatment is the possibility that an immune reaction is triggered against the pharmaceutical preparation because, due to its molecular size, it can be recognized by the patient's immune system. The appearance of antibodies against botulin A is a serious problem since it contributes to a clear loss of efficacy of the treatment (6-13). This loss of efficacy of BOTOX® treatment entails the need to increase the concentration of the preparation in subsequent treatments, which in turn produces an enhancement of the immune response. As an alternative, the use of serotypes other than botulinum toxins (BoTox), such as BoTox B, BoTox F and BoTox E. However, the application of pharmaceutical preparations with different serotypes can not be considered a solution to the problem since, late or • early, the immune reaction can reoccur. In addition, the treatment with botulinum toxins is expensive due, mainly, to the lability and instability of the pharmaceutical preparations containing them. There is, therefore, an imperative need to develop molecules that mimic the paralytic effects of botulinum toxins but that are endowed with much simpler and more stable molecular structures, which do not induce immune reactions, and whose production cost is economical. Molecules of a peptide nature fulfill these properties.

SUBSTITUTE SHEET (RULE 26) Amino acid sequences that inhibit neuronal exocytosis have been described. In particular, it has been shown that peptides with a number of amino acids greater than 20, derived from the C-terminal sequence of SNAP-25, block the release of catecholamines from permeabilized chromaffin cells (14). In the same way, peptides have been described-derived from the amino acid sequences of the proteins syntaxin and VAMP that can also affect the exocytotic process (15). Although these peptides show biological activity, their further development as cosmetic and / or therapeutic agents has not occurred, probably due to their size since they make their development and difficult as useful therapeutic agents difficult and expensive. Therefore, there is a need to find molecules of smaller size that can be applied in cosmetics and medicine. This invention provides a solution to existing needs comprising the discovery of smaller amino acid sequences, between 3 and 30 amino acids, derived from the amino terminus (N-terminal domain) of the SNAP-25 protein, which inhibit neuronal exocytosis. Furthermore, it has been discovered that the simultaneous use of peptides derived from the amino terminus and the carboxyl end (C-terminal domain) of SNAP-25 produces a considerable increase in its inhibitory activity, that is, there is an enhancement of its activity against the shown by the individual peptides. Therefore, an object of this invention is a peptide having a sequence consisting of 3 to 30 contiguous amino acids contained in the amino terminus of the protein SNAP-25, which inhibits neuronal exocytosis. A further object of this invention is a nucleic acid which essentially encodes a peptide of. those provided by this invention. Plasmids and vectors containing said nucleic acid (also identified as SUBSTITUTE SHEET (RULE 26) constructs), as well as cells transformed with said plasmids or vectors expressing a peptide of the invention also constitute additional objects of the present invention. Another additional object of this invention is a mixture of at least one peptide of those provided by this invention and at least one peptide having a sequence consisting of 3 to 30 contiguous amino acids contained in the carboxyl terminus of the protein. SNAP-25. Another additional object of this invention is a cosmetic composition comprising at least one peptide of those provided by this invention. The use of the peptides provided by this invention in the preparation of said cosmetic composition, as well as the method of cosmetic treatment comprising the application of said cosmetic composition constitute additional objects of this invention. Another additional object of this invention is a pharmaceutical composition comprising at least one peptide of those provided by this invention, or, alternatively, a vector containing a nucleic acid encoding a peptide of the invention. The use of peptides and vectors (constructions) provided by this invention in the manufacture of said pharmaceutical compositions, as well as the method of treatment of the human or animal body comprising the application of said cosmetic composition constitute additional objects of this invention. Another additional object of the present invention is a combination of drugs comprising at least one peptide of those provided by this invention together with at least one drug intended for a second therapeutic target which may be the same as or different from the therapeutic target to which the peptide provided by SUBSTITUTE SHEET (RULE 26) this invention.

DETAILED DESCRIPTION OF THE INVENTION This invention provides a peptide derived from the amino terminus of the SNAP-25 protein. More specifically, the invention provides a peptide, hereinafter the peptide of the invention, having a sequence of 3 to 30 contiguous amino acids contained in SEQ. ID. N °: 1 [see the section relating to the LIST OF SEQUENCES]. The invention also includes peptides substantially homologous to the peptide of the invention. In the sense used in this description, the term "substantially homologous" means that the peptide in question has a homology at the amino acid level of at least 60%, preferably at least 80%, and, more preferably, , of, at least, 95%. The invention also includes peptides functionally equivalent to the peptide of the invention. In the sense used in this description, the term "functionally equivalent" means that the peptide in question has, at least, one of the biological activities of the peptide of the invention, for example, the ability to inhibit, at least partially, the exocytosis. neuronal In a particular embodiment, the peptide of the invention has a length of 3 to 20 amino acids, preferably, 6 to 19 amino acids. The amino acids constituting the structural units of the peptide of the invention may have the D or L configuration. The amino terminus of the amino terminus may have the acetylated terminal amino group and the amino acid of the carboxyl terminus may have the carboxyl terminal amidated group. Therefore, the present invention also includes derivatives of the peptide of the invention in which the end SUBSTITUTE SHEET (RULE 26) amino terminal is acetylated and / or in which the carboxyl terminal end is amidated. Particular examples of peptides of the invention are peptides having the amino acid sequences shown in SEQ. ID. Nß: 2 and SEC. ID. No. 3. Within the scope of this invention are the cosmetically and / or pharmaceutically acceptable salts of the peptide of the invention. The term "cosmetically and / or pharmaceutically acceptable salts" includes the salts usually used to form metal salts or addition salts of free acids or bases. The nature of the salt is not critical, as long as it is cosmetic and / or pharmaceutically acceptable. The cosmetically and / or pharmaceutically acceptable salts of the peptide of the invention can be obtained from acids or bases, organic or inorganic, by conventional methods well known to those skilled in the art by reacting the appropriate acid or base with the peptide of the invention. invention. Additionally, the peptide of the invention can undergo reversible chemical modifications in order to increase its bioavailability (including stability and liposolubility) and its ease of passage of the blood-brain barrier and epithelial tissue. Examples of such reversible chemical modifications include the esterification of the carboxylate groups of the glutamic and aspartic amino acids with an acetomethyl group, whereby the negative charge of the amino acid is eliminated and its hydrophobicity is increased. This esterification is reversible since the ester bond formed is recognized by intracellular esterases that hydrolyze it, returning the charge to the aspartic and glutamic residues. The net effect is an accumulation of intracellular peptide since the internalized and de-esterified peptide can not cross the membrane SUBSTITUTE SHEET (RULE 26) cellular. The peptide of the invention can be obtained by conventional methods of chemical synthesis of solid phase peptides following the methodology based on Fmoc and / or Boc (16). Alternatively, the peptide of the invention can be obtained by conventional methods based on recombinant DNA technology, for example, by a method that, briefly, comprises inserting the nucleic acid sequence encoding the peptide of the invention into a plasmid or vector appropriate, transforming competent cells for said plasmid or vector, and growing said cells under conditions that allow the expression of the peptide of the invention and, if desired, isolate and, optionally, purify the peptide of the invention by conventional methods known to those skilled in the art. The matter. The nucleic acid sequence encoding the peptide of the invention can be easily deduced from the correspondence between the amino acids and the codons of nucleotides encoding such amino acids. In this case, an additional object of this invention is an isolated nucleic acid sequence encoding the peptide of the invention. In a particular embodiment, said nucleic acid is selected from single-stranded DNA, double-stranded DNA and RNA. A further object of this invention is the plasmids and expression vectors containing said nucleic acid sequence encoding the peptide of the invention, as well as prokaryotic or eukaryotic cells expressing the peptide of the invention. A review of the principles of recombinant DNA technology can be found, for example, in the textbook entitled ^ Principles of Gene Manipulation: An Introduction to Genetic Engineering, R.W.; S.B. Primrose, edited by Bláckwell Scientific Publications, 4 * edition (1989).

SUBSTITUTE SHEET (RULE 26) The peptide of the invention is capable of inhibiting, at least partially, the neuronal exocytosis, probably by a mechanism that involves interference in the assembly of the complex or fusion protein (SNARE) and / or its destabilization thermal The ability of the peptides of the invention as inhibitors of neuronal exocytosis (neurosecretion) was demonstrated by an assay that evaluates the potency of said peptides in inhibiting the release of calcium-induced catecholamines in chromaffin cells permeabilized with a detergent [ see Example 1.2.1]. Briefly, the chromaffin cells in culture are incubated with adrenaline and noradrenaline labeled with tritium, permeabilized with digitonin, stimulated with calcium and the amount of radioactivity released by the cells is measured to the extracellular medium, which is a reflection of the exocytosis of said cells. marked catecholamines. The hexapeptide of the invention [SEQ. ID. N °: 2], at a concentration of 1 mM, blocked approximately 20% of the release of catecholamines (adrenaline and noradrenaline) in permeabilized chromaffin cells, while the 13 amino acid peptide [SEC. ID. No. 3], at a concentration of 1 mM, inhibited about 35% of the release of catecholamines in the permeabilized chromaffin cells. The peptides shown in SEQ. ID. No. 5 and SEC. ID. Nß: 6, from the carboxyl end of SNAP-25 [SEC. ID. Nß: 4] inhibited Ca2"-induced secretion in chromaffin cells permeabilized with digitonin by approximately 40% when used at a concentration of 1 mM Parallel assays performed using, together, at least one peptide from the amino terminus of the SNAP. -25, for example, the peptide of SEQ ID NO-2 or SEQ.

SUBSTITUTE SHEET (RULE 26) ID. No. 3, and at least one peptide from the carboxyl terminus of SNAP-25, for example, the peptide of SEQ. ID. Nc: 5 or SEC. ID. N °: 6, showed that the joint use of, at least, a peptide from the amino end of SNAP-25 and, at least, a peptide from the carboxyl end of SNAP-25, potentiates the observed biological activity for each of the peptides tested separately. In a particular case, mixtures of peptides consisting of one of the peptides shown in SEQ. ID. Nß: 2 or in the SEC. ID. Nc: 3 and one of the peptides shown in SEC. ID. Nß: 5 or in the SEC. ID. No. 6, at a concentration of 0.5 mM each, obtaining a percentage of inhibition of release of catecholamines in permeabilized chromaffin cells of 55%. Taken together, these results indicate that both types of peptides, both those from the amino terminus and those from the carboxyl terminus, inhibit the exocytosis of catecholamines, and that the joint use of peptides from the amino terminus and the carboxyl end enhances the activity observed for each of them separately. Therefore, the invention further provides a mixture of peptides comprising: (a) at least one peptide of the invention, and (b) at least one peptide having a sequence of from 3 to 30 contiguous amino acids contained in the SEC . ID. Nß: 4 [hereinafter, peptide (COOH) to indicate its relationship with the carboxyl terminus of SNAP-25]. In a particular embodiment, said mixture of peptides is constituted by, at least, a peptide selected from the group formed by the peptides shown in SEQ. ID. N ": 2 and in SEQ ID NO: 3, and at least one selected peptide SUBSTITUTE SHEET (RULE 26) of the group formed by the peptides shown in SEC. ID. Nß: 5 and in the SEC. ID. Nß: 6. The ability of the peptides of the invention to interfere with the formation and stability of the fusion complex (SNARE) was demonstrated by carrying out reconstitution tests in vi tro of the protein fusion complex with proteins recombinants [see Example 1.2.2]. Briefly, the SNAP-25 protein was immobilized in 96-well plates, the VAMP and syntaxin proteins were added in the presence and / or absence of the peptides of the invention and the formation of the fusion protein complex (SNARE) was evaluated. Detection of the complex is performed using an antibody against syntaxin (anti-syntaxin), followed by a second labeled antibody that recognizes the anti-syntaxin antibody. The obtained data seem to indicate that the presence of the peptides of the invention during the assembly of the fusion complex produces a significant decrease thereof. Therefore, the mechanism of the inhibitory action of neuronal exocytosis seems to imply that the peptides of the invention interfere in the formation and / or stability of the fusion protein complex (SNARE). The results obtained with said tests suggest that the peptides of the invention, small peptides, between 3 and 30 amino acids, derived from the amino acid sequence of the amino terminus of SNAP-25, optionally-together with peptides from the carboxyl terminus of SNAP-25, act as inhibitors of neuronal exocytosis. Since these peptides mimic the sequences of neuronal proteins involved in exocytosis, their activity is specific since they only interact with the corresponding neuronal proteins without affecting other cellular components. The mechanism of action of the peptides of the invention SUBSTITUTE SHEET (RULE 26) appears to be similar to that of botulinum toxins, thus affecting the formation and / or stability of the fusion protein complex, so it can be considered that the peptides of the invention have cosmetic applications. Therapeutics similar to those described for botulinum toxin. Therefore, the peptides of the invention can be converted into effective, stable, safe and economical substitutes for botulinum toxins both in the treatment of facial wrinkles and / or facial asymmetry and in the treatment of the symptomatology of spasmodic diseases, and their use as neuroprotectors in the treatment of neurological disorders and neurodegenerative diseases. The studies carried out by the applicants also suggest the innovative concept of the simultaneous use of peptides from the N-terminal and C-terminal domains of SNAP-25 as modulators of neuronal exocytosis. As a whole, the results obtained with the peptides of the invention, together with their stability and structural simplicity and the chemical diversity that can be obtained based on the composition of the amino and carboxyl ends of SNAP-25, confer to the peptides of the invention a high cosmetic and / or therapeutic potential. The peptides of the invention can be used for cosmetic and / or therapeutic purposes mediated by a pathological neuronal exocytosis. Among the cosmetic applications of the peptides of the invention are the treatment and elimination, total or partial, of facial wrinkles and / or facial asymmetry in a human being. The invention provides a cosmetic composition comprising a cosmetically effective amount of at least one peptide of the invention together with at least one adjuvant SUBSTITUTE SHEET (RULE 26) cosmetically acceptable. Additionally and optionally, said cosmetic composition may contain one of the peptides identified as peptide (COOH). For their cosmetic applications, the peptides of the invention can be applied by any means that produces the contact of the peptide with the site of action thereof in the body of a mammal, preferably the human being. The amount of cosmetically effective peptide to be applied as well as its dosage for the treatment of facial wrinkles and / or facial asymmetry with the peptides and / or cosmetic compositions of the invention will depend on numerous factors, including age, condition of the subject who wants the treatment, the severity of the wrinkles and / or the facial asymmetry, the route and frequency of application and the particular peptide to be used. The cosmetic compositions containing the peptides of the invention can be presented in any suitable application form, for example, solid, liquid or semisolid, such as creams, ointments, gels or solutions, and can be applied by any appropriate route, preferably, via topical, for which they will include the cosmetically acceptable adjuvants necessary for the formulation of the desired form of administration. In a particular and preferred embodiment, the peptides of the invention are encapsulated in liposomes, optionally together with one or more other peptides (COOH), which are incorporated into the other components of the cosmetic preparation. A review of the various cosmetic forms of application of active compounds and of the adjuvants necessary for obtaining them can be found, for example, in the textbook "Cosmetology of Harry", Wilkinson & Moore, Ed. Díaz de Santos (1990). Therefore, a further object of this invention SUBSTITUTE SHEET (RULE 26) constitutes the use of the peptides of the invention in the preparation of cosmetic compositions for the treatment of facial wrinkles and / or facial asymmetry. The invention also provides a method for the cosmetic treatment in a mammal, preferably in humans, of facial wrinkles and / or facial asymmetry which comprises applying to said mammal having facial wrinkles and / or facial asymmetry., a cosmetically effective amount of at least one peptide of the invention, optionally together with one or more peptides (COOH), preferably in the form of a cosmetic composition containing it. Additionally, the peptides of the invention are suitable for the treatment of spasmodic diseases, for example, dystonia, strabismus, blepharospasm, torticollis, tics, etc.; and / or as neuroprotectants in the treatment of neurological disorders and / or neurodegenerative diseases. Among such neurological disorders are acute neurological complaints, for example, those that occur in the early stages of cerebral ischemia. It is known that during an ischemic process an uncontrolled release occurs in the affected area of the neurotransmitter glutamate. This neurotransmitter interacts with specific neuronal membrane receptors, causing a massive influx of calcium ions into the neuron. The intracellular calcium causes the release of more glutamate, thus triggering a chain reaction. In addition, the massive and prolonged entry of calcium into neurons causes their death which results in the appearance of necrotic tissue in the ischemic area. Clearly, the progress of ischemic damage can be stopped, at least partially, if the uncontrolled exocytosis of glutamate is controlled. Therefore, the peptides of the invention, by their SUBSTITUTE SHEET (RULE 26) ability to inhibit exocytosis may be adequate to prevent and / or slow neuronal death characteristic of the ischemic process, so they are useful in the treatment of neuropathologies that occur due to an excessive exocytosis of glutamate as, for example, example, senile dementia, Alzheimer's dementia, dementia associated with AIDS, epilepsy, amyotrophic sclerosis, multiple lateral sclerosis, etc. In this case, the application in the treatment of neurological diseases would be similar to that described for botulinum toxin A (18). The peptides of the invention could therefore be part of a combination therapy (directed to several therapeutic targets) whose objective was to more effectively stop neurodegeneration. A further object of this invention is a pharmaceutical composition comprising a therapeutically effective amount of at least one peptide of the invention together with at least one pharmaceutically acceptable excipient. In a particular embodiment, said pharmaceutical composition further contains one or more peptides (COOH). Alternatively, the pharmaceutical composition of the invention may contain a therapeutically effective amount of a vector that contains at least one nucleic acid sequence encoding a peptide of the invention, together with at least one adjuvant and / or a pharmaceutically acceptable excipient. Said vector can be used in gene therapy. The active products of the invention (peptides or vectors) can be administered for the treatment of pathological neuronal exocytosis, manifested, for example, by spasmodic diseases, neurological disorders or neurodegenerative diseases, by any means that produces the contact of the peptide with the site of action thereof in the body of a mammal, preferably the human being.

SUBSTITUTE SHEET (RULE 26) The amount of active product of the invention [peptides or vectors (constructs)] therapeutically effective to be administered as well as its dosage to treat a pathological state with the peptides and / or pharmaceutical compositions of the invention will depend on numerous factors, including the age, condition of the patient, the severity of the disorder or disorder, the route and frequency of administration and the particular peptide to be used. The pharmaceutical compositions containing the peptides or the vectors (constructs) of the invention can be presented in any form of appropriate administration, for example, solid, liquid or semi-solid, such as ointments, creams, gels or solutions, and can be administered by any route appropriate, for example, orally, parenterally or topically, for which purpose they will include the pharmaceutically acceptable excipients necessary for the formulation of the desired administration form. A review of the different pharmaceutical forms of administration of drugs and of the excipients necessary to obtain them can be found, for example, in the "Galenic Pharmacy Treaty", C. Faulí i Trillo, 1993, Luzán 5, S.A. Editions, Madrid. As previously mentioned, the peptides of the invention could be part of a combination therapy in order to slow down neurodegeneration more effectively. In this case, the invention provides a pharmaceutical composition comprising, at least one peptide of the invention, optionally together with another or other neuronal exocytosis inhibitor compounds, and together with, at least, one drug intended for another therapeutic target, selected from the group consisting of a neuronal glutamate receptor blocker, a calcium chelating agent, an antioxidant, a free radical scavenger and their mixtures.

SUBSTITUTE SHEET (RULE 26) In a particular embodiment, said composition useful in a combination therapy can contain at least one peptide of the invention, optionally together with another or other compounds capable of inhibiting neuronal exocytosis and a receptor blocker of neuronal glutamate. In another embodiment of this invention said composition could contain, at least one peptide of the invention, optionally together another or other compounds capable of inhibiting neuronal exocytosis, a neuronal glutamate receptor blocker, a calcium chelating agent, an antioxidant and / or a free radical scavenger. Among the compounds capable of inhibiting neuronal exocytosis are the peptides from the carboxyl terminus of SNAP-25 identified as peptides (COOH). Many other examples of compositions can be proposed, all having in common the need to control the exocytosis of neurotransmitters. A further object of this invention is the use of the peptides of the invention or of vectors containing at least one sequence encoding a peptides of the invention, in the preparation of a medicament for the treatment of diseases and / or pathological alterations mediated by pathological neuronal exocytosis, for example, spasmodic diseases, neurological disorders and / or neurodegenerative diseases. The invention further provides a method for the treatment in a mammal of diseases and pathological alterations mediated by pathological neuronal exocytosis, for example, spasmodic diseases, neurological disorders and / or neurodegenerative diseases, which comprises administering to said mammal suffering from said disease or pathological alteration a therapeutically effective amount of at least one peptide of the invention, or of a vector containing at least one sequenceSUBSTITUTE SHEET (RULE 26) of DNA encoding a peptide of the invention, preferably, in the form of a pharmaceutical composition containing it. In a particular embodiment of this invention said pharmaceutical composition contains, in addition to the peptide or peptides of the invention, one or more peptides (COOH). The following examples serve to illustrate the nature of the present invention and should not be considered in a limiting sense thereof.

EXAMPLE 1 Inhibitor peptides of neurotransmitter exocytosis 1.1 Synthesis of peptides The peptides shown in SEQ. ID. N: 2, SEC. ID. No. 3, SEC. ID. No. 5 and SEC. ID. N °: 6 by conventional methods of synthesis of peptides in solid phase using the synthetic methodology based on Fmoc and / or Boc (16). The resulting peptides were purified by high performance liquid chromatography (HPLC) and analyzed by mass spectrometry. 1. 2 Evaluation of biological activity To evaluate the biological activity of the peptides obtained in Example 1.1, an assay was developed which evaluates the potency of said peptides in the inhibition of the calcium-induced release of catecholamines in chromaffin cells and a reconstitution assay in vi of the fusion complex (SNARE). 1. 2.1 Inhibition of the release of catecholamines This test was performed to test the ability of the peptides synthesized in Example 1.1 as inhibitors of neuronal exocytosis. In this test, the potency of said peptides in the inhibition of the release of SUBSTITUTE SHEET (RULE 26) Catecholamines (noradrenaline and adrenaline) induced by calcium in chromaffin cells (obtained from bovine adrenal glands) permeabilized with the detergent digitonin, according to the method described by Gutiérrez et al. (1995 and 1997). Briefly, the chromaffin cells in culture are incubated with [3 H] -adrenaline and [3 H] -noradrenaline, permeabilized with 20 μM digitonin, and stimulated with calcium (10 μM), in the presence of the peptides to be tested (separated or mixed) ), and the amount of radioactivity released by the cells into the extracellular medium is measured, which is a reflection of the exocytosis of [3 H] -adrenaline and [3 H] -noradrenaline. The results obtained in the inhibition of the release of catecholamines in permeabilized chromaffin cells were the following: a) the peptide of SEC. ID. No. 2, from the amino terminus of SNAP-25, at a concentration of 1 mM, blocked approximately 20% of the release of catecholamines in permeabilized chromaffin cells; b) the peptide of SEC. ID. Nc: 3, from the amino end of SNAP-25, at a concentration of 1 mM, inhibited approximately 35% of the release of catecholamines in permeabilized chromaffin cells; c) the peptides of SEC. ID. No. 5 and SEC. ID. Nß: 6, from the carboxyl end of SNAP-25, at a concentration of 1 mM, inhibited Cas * -induced secretion in chromaffin cells permeabilized with digitonin by approximately 40%; and d) mixtures of peptides consisting of one of the peptides shown in SEQ. ID. No.: 2 or in the SEC. ID. Nß: 3 and one of the peptides shown in SEC. ID. No. 5 or in the SEC. ID. Nß: 6, at a concentration of 0.5 mM of each of them, inhibited the release of catecholamines in permeabilized chromaffin cells by approximately 55%.

SUBSTITUTE SHEET (RULE 26) In this set, these results indicate that both types of peptides, both those from the amino terminus and those from the carboxyl end, inhibit the exocytosis of catecholamines, and that the joint use of peptides from the amino terminus and the carboxyl end enhances the biological activity observed for each of them separately. 1. 2.2 In vitro reconstitution This test was performed to determine the ability of the peptides obtained in Example 1.1 to interfere with the formation and stability of the fusion complex (SNARE). The test consists of evaluating the in vitro reconstitution of the fusion protein complex with recombinant proteins produced in Escherichia coli. Reconstitution assays based on ELISA methods (Enzyme-Linked Immuno Assay), involve the immobilization of the SNAP-25 protein in 96 well plates and the subsequent formation of the fusion protein complex by adding the VAMP and syntaxin proteins in the presence and / or absence of the Iz peptides. invention. The detection of the complex is carried out using an antibody against the protein syntaxin (anti-syntaxin), followed by an antibody that recognizes the anti-syntaxin antibody covalently labeled with a peroxidase. The amount of fusion protein complex was followed by adding 1,2-phenylenediamine dichloride whose reaction with peroxidase causes a product with yellowish-orange color that absorbs at 492 nm in an acid medium. The data obtained showed that the presence of the peptides obtained in Example 1.1 during the assembly of the fusion complex produces a significant decrease thereof. Therefore, the mechanism of action of said peptides seems to involve the interference of such SUBSTITUTE SHEET (RULE 26) peptides in the formation and / or stability of the fusion protein complex (SNARE).

BIBLIOGRAPHY 1. Schiavo, G., Rossetto O. and Montecucco C. Molecular bases of tetanus and botulism. Research and Science 234, 46-55. 2. Montecucco, C. and Schiavo, G. (1994). Mechanism of action of tetanus and botulinum neurotoxins. Mol. Microbiol. 13, 1-8. 3. Schiavo, G., Rosetto, O., Benfenati, F., Poulain, B. and Montecucco, C. (1994). Tetanus and botulinum neurotoxins are zinc proteases specific for components of the neuroexocytosis apparatus. Ann. NY Acad. Sci. 710, 65-75. 4. Calakos, N. and Scheller, R.H. (nineteen ninety six). Synaptic vesicle biogenesis, docking and fusion: a molecular description. Physiol. Rev. 76, 1-29. 5. Sutton, R.B., Fasshauer, D., Jahn, R. and Brunger, A.T. (1998). Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4A resolution. Nature 395, 347-353. 6. Jankovic, J. and Brin, F.M. (1991). Therapeutic uses of botulinum toxin. New Engl. J. Med. 324.1186-1194. 7. Jankovic, J. (1994). Botulinum toxin in movement disorders. Curr. Opin. Neurol. 6, 358-366. 8. Jankovic J. and Brin M.F. (1997). Botulinum toxin: historical perspective and potential new indications. Muscle Nerve Suppl. 6, S129-S145. 9. Davis, L.E. (1993). Botulinum toxin-from poison to medicine. West J. Med, 128, 25-28. 10: Hughes, A.J. (1994). Botulinum toxin in clinical practice. Drugs 48, 888-893.

SUBSTITUTE SHEET (RULE 26) 11. Hambleton, P. (1992). Clostridium botulinum toxins a general review of involvement in disease, structure, mode of action and preparation for clinical use. J. Neurol. 239, 16-20. 12. Borodic, G.E. and Pearces, L.B. (1994). New concepts in botulinum toxin therapy. Drug Safety 11, 145-152. 13. Brin, M.F., Blitzer, A., Stewart, C, Pine, Z., Borg-Stein, J., Miller J., Nagalapura, N.S., and Rosenfeld, D.B. (1993). Disorders with excessive muscle contraction: Candidates for treatment with intramuscular botulinum toxin ("BoTox"). Botulinum and Tetanus Neurotoxins (Ed. B.R. DasGupata), 559-576. 14. Gutiérrez, L.M. , Canaves J., Ferrer-Montiel, A.V. , Reig, J.A., Montal, M., and Viniegra, S. (1995). A peptide that mimics the carboxy terminal domain of SNAP25 blocks Ca2 + - dependent exocytosis in chromaffin cells. FEBS Lett 372, 39-43. 15. Augine, G.J., Burns, M.E., DeBello W.M. and Schweizer, F.E. (nineteen ninety six). Exocytosis: Proteins and disturbations. Annu. .Rev. Pharmacol. Toxi col. 36, 659-701. 16. Pennington, M.W. and Dunn, B.N. (1994). Peptide synthesis protocols. Humana Press, Totowa. 17. Gutiérrez, L.M. , Viniegra, S., Rueda, J., Ferrer-Montiel, A.V., Canaves, J.M. and Montal. M. (1997). A peptide that mimics the C-terminal sequence of SNAP-25 inhibits secretory vesicle docking in chromaffin cells. J. Biol. Chem. 272, 2634-2639. 8. Clarke, CE. (1992). Therapeutic potential of botulinum toxin in neurological disorders. Quart. J. Med. 299, 197-205.

SUBSTITUTE SHEET (RULE 26) LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: UNIVERSIDAD MIGUEL HERNÁNDEZ (B) STREET: Monóvar s / n (C) CITY: Elche (D) PROVINCE: Alicante (E) COUNTRY: ES (F) CODE POSTAL (ZIP): 03206 (G) TELEPHONE: 96 665 8727 (H) FAX: 96 665 8680 (ii) TITLE OF THE INVENTION: PEPTIDOS INHIBITORS OF THE NEURONAL EXOCYTOSIS, COSMETIC AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM (iii) SEQUENCE NUMBER: 6 (iv) LEGIBLE FORMAT BY COMPUTER: (A) TYPE OF SUPPORT: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (OEP) (2) INFORMATION OF THE SEC. ID. Nß: 1 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 82 amino acids (B) TYPE: amino acid (C) NUMBER OF CHAINS: simple (D) TOPOLOGY: linear - SUBSTITUTE SHEET (RULE 26) (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. No.: 1: Wing Glu Asp Wing Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg Arg 1 '5 10 15 Wing Asp Gln Leu Wing Asp Glu Being Leu Glu Being Thr Arg Arg Met Leu 20 25 30 Gln Leu Val Glu Glu Ser Lys Asp Ala lie Arg Thr Leu Val Met Leu 35 40 45 Asp Glu Gln Gly Glu Gln Leu Glu Arg lie Glu Glu Gly Met Asp Gln 50 55 60 lie Asn Lys Asp Met Lys Glu Wing Glu Lys Asn Leu Thr Asp Leu Gly 65 70 75 80 Lys Phe (2) INFORMATION OF THE SEC. ID. No.: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) NUMBER OF CHAINS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Peptide SUBSTITUTE SHEET (RULE 26) (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. No.: 2: Glu Glu Met Gln Arg Arg 1 5 (2) .INFORMATION OF SEC. ID. N °: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) NUMBER OF CHAINS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Peptide (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. ID. Nß: 3: Glu Leu Glu Glu Met Gln Arg Arg Ala Asp Gln Leu Ala 1 5 10 (2) INFORMATION OF THE SEC. ID. Nß: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 86 amino acids (B) TYPE: amino acid (C) NUMBER OF CHAINS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. Nß: 4: Val Asp Glu Arg Glu Gln Met Wing He Ser Gly Gly Phe He Arg Arg 1 5 10 15 Val Thr Asn Ala_Arg Glu Asn Glu Glu Met Asp Glu Asn Leu Glu Gln 20 25 30 SUBSTITUTE SHEET (RULE 26) Val Ser Gly He Leu Gly Asn Leu Arg His Met Ala Leu Asp Met Gly 35 40 45 Asn Glu He Asp Thr Gln Asn Arg Gln He Asp Arg He Met Glu Lys, 50 55 60 Wing Asp Ser Asn Lys Thr Arg He Asp Glu Wing Asn Gln Arg Wing Thr 65 70 75 80 Lys Met Leu Gly Ser Gly 85 (2) INFORMATION OF THE SEC. ID. N °: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) NUMBER OF CHAINS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Peptide (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. ID. Nß: 5: Arg He Met Glu Lys Wing Asp Ser Asn Lys Thr Arg He Asp Glu Wing 1 5 10 15 Asn Gln (2) INFORMATION OF THE SEC. ID. N °: 6: (i) CHARACTERISTICS OF THE SEQUENCE: SUBSTITUTE SHEET (RULE 26) (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) NUMBER OF CHAINS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: Peptide '(xi) DESCRIPTION OF THE SEQUENCE : SEC. ID. N °: 6: Wing Asp Ser Asn Lys Thr Arg He Asp Glu Wing Asn Gln Arg Wing Thr 1 5 10 15 Lys Met Le SUBSTITUTE SHEET (RULE 26)

Claims

1. A peptide characterized in that it has a sequence of 3 to 30 contiguous amino acids contained in SEQ. ID. No.: 1

2. The peptide according to claim 1, characterized in that it is substantially homologous to a peptide having a sequence of 3 to 30 contiguous amino acids contained in SEQ. ID. Nß: 1.

3. Peptide functionally equivalent to a peptide according to claim 1 or 2, characterized in that it is capable of inhibiting, at least partially, the neuronal exocytosis.

4. The peptide according to claim 1, characterized in that it has a length of from 3 to 20 amino acids, preferably from 6 to 19 amino acids.

5. Peptide according to claim 1, characterized in that said amino acids have the configuration D.

6. The peptide according to claim 1, characterized in that said amino acids have the L configuration.

7. The peptide according to claim 1, characterized in that the amino terminal amino acid has the acetylated terminal amino group.

8. The peptide according to claim 1, characterized in that the carboxyl-terminal amino acid has the amidated terminal carboxyl group.

SUBSTITUTE SHEET (RULE 26) 24 9. Peptide according to claim 1, characterized in that it has an amino acid sequence selected from the amino acid sequences shown in SEQ. ID. Nß: 2 and SEC. ID. Nß: 3

10. The peptide according to claim 1, characterized in that it also contains a reversible modification in order to increase its bioavailability and ease of passage of the blood-brain barrier and epithelial tissue.

11. An isolated nucleic acid sequence characterized in that it encodes a peptide according to any of claims 1 to 10.

12. Nucleic acid sequence according to claim 11, characterized in that said nucleic acid is selected from double-stranded DNA, single-stranded DNA and RNA.

13. A plasmid characterized in that it contains a nucleic acid sequence according to claim 11.

14. An expression vector characterized in that it contains a nucleic acid sequence according to claim 11.

. 15. A prokaryotic or eukaryotic cell characterized in that it expresses a peptide according to any of claims 1 to 10.

16. A mixture of peptides characterized in that it is formed by: (a) at least one peptide according to any of claims 1 to 10, and (b) at least one peptide having a sequence of 3 to SUBSTITUTE SHEET (RULE 26) 25 30 contiguous amino acids contained in SEC. ID. No.: 4

17. Mixture according to claim 16, characterized in that it is formed by: (a) at least one peptide selected from the group consisting of the peptides shown in SEQ. ID. No.: 2 and in the SEC. ID.'N ": 3, and (b) at least one peptide selected from the group consisting of the peptides shown in SEQ ID NO: 5 and SEQ ID N: 6.

18. A cosmetic composition comprising a cosmetically effective amount of at least one peptide according to any of claims 1 to 10, together with at least one cosmetically acceptable adjuvant.

19. Cosmetic composition according to the re-excitation 18, optionally comprising, in addition, at least one peptide having a sequence of 3 to 30 contiguous amino acids contained in SEQ. ID. Nß: 4

20. Use of a peptide according to any of claims 1 to 10, in the preparation of a cosmetic composition for the treatment of facial wrinkles and / or facial asymmetry.

21. A method for the cosmetic treatment in a human being of facial wrinkles and / or facial asymmetry that comprises applying to said human being that has facial wrinkles and / or facial asymmetry, a cosmetically effective amount of, at least, one peptide according to any of claims 1 to 10, optionally together with one or more peptides having a sequence of 3 to 30 amino acids SUBSTITUTE SHEET (RULE 26) 26 contiguous contents in the SEC. ID. Nß: 4, preferably in the form of a cosmetic composition according to any of claims 18 or 19.

22. A pharmaceutical composition comprising a therapeutically effective amount of at least one peptide according to any of claims 1 to 10, together with at least one pharmaceutically acceptable excipient.

23. The pharmaceutical composition of claim 22, further comprising, optionally, one or more peptides having a sequence of from 3 to 30 contiguous amino acids contained in SEQ. ID. No.: 4

24. The pharmaceutical composition according to claim 22, optionally further comprising a drug selected from the group consisting of a neuronal glutamate receptor blocker, a calcium chelating agent, an antioxidant, a free radical scavenger and mixtures thereof, and optionally , another or other inhibitory blocks of neuronal exocytosis.

25. Composition according to claim 24, which optionally further comprises one or more peptides having a sequence of 3 to 30 contiguous amino acids contained in SEQ. ID. Nß: 4

26. A pharmaceutical composition comprising a therapeutically effective amount of a vector containing at least one nucleic acid sequence according to claim 11, encoding a peptide according to any of claims 1 to 10, together with at least one adjuvant and / or a pharmaceutically acceptable excipient.

SUBSTITUTE SHEET (RULE 26) 27 27. Use of a peptide according to any of claims 1 to 10 in the preparation of a medicament for the treatment of diseases and / or pathological changes mediated by pathological neuronal exocytosis.

28. Use of a vector containing at least one nucleic acid sequence according to claim 11, which encodes a peptide according to any of claims 1 to 10, in the preparation of a medicament for the treatment of diseases and / or pathological alterations mediated by pathological neuronal exocytosis. SUBSTITUTE SHEET (RULE 26)