Peptides inhibiting neuron exocytosis

FIELD: medicine.

SUBSTANCE: invention refers to peptides of general formula Rr-AA-R2 which can regulate neuron exocytosis, their mixtures and their cosmetically and pharmaceutically acceptable salts wherein AA represents a sequence of 6-40 neighbouring amino acids contained in the amino acid sequence of the protein SNAP-25 R1 is specified in a group consisting of H or acetyl, or saturated or unsaturated, linear, branched or cyclic C3-C24acyl, or a polyethylene glycol polymer; R2 is specified in a group consisting of amino, unsubstituted or substituted by saturated linear or branched, or cyclic C2-C24aliphatic groups provided when R1 represents H or acetyl, R2 is other tuhan unsubstituted amino. The invention also refers to cosmetic or pharmaceutical compositions containing such peptides, and use of such for treating the conditions which require regulation of neuron exocytosis, preferentially for skin treatment.

EFFECT: higher clinical effectiveness.

12 cl, 1 dwg, 8 ex

 

The SCOPE of the INVENTION

The present invention relates to peptides that can regulate neuronal exocytosis, and to cosmetic or pharmaceutical compositions containing these peptides, suitable for treatment of conditions requiring regulation of neuronal exocytosis, such as, for example, muscle spasticity, facial asymmetry and/or wrinkles on the face, preferably wrinkles.

PRIOR art

One of the most visible signs of human aging are changes that affect the skin: dryness, appearance of spots, dullness and wrinkles. These effects can be caused by external agents, such as, for example, constant exposure to sunlight, atmospheric pollution or contact with chemical agents that are present in cleansing products and also they are the result of internal physiological, biochemical and histological changes in the human body associated with reduced synthesis of proteins, such as collagen or elastin, increased proteolysis, and the total destruction of the skin barrier, connective tissue and cohesion.

Different active ingredients have been described to prevent and reduce the symptoms of aging, such as, for example, retinoids, hydroxy acids, flavonoids or derivatives of vitamins C and E. These joint is usually operate, improving the hydration of the skin, increasing cell renewal or preventing degeneration of tissue that forms the skin;

however, their effectiveness in the prevention and treatment of facial wrinkles caused by muscle contraction is limited. The basis or mechanism of the appearance of facial wrinkles on the face is the voltage epidermal muscles within the skin. This muscle tension is a result of overactive nerves that Innervate the facial muscles. Hyperactivity of nerves characterized by uncontrolled and excessive release of neurotransmitters, excitatory muscle fibers. On the other hand, molecules that regulate neuronal exocytosis, promote relaxation of muscle tension and, accordingly, elimination of facial wrinkles.

There is therefore a need to develop new active ingredients with proven efficacy for the production of cosmetic or pharmaceutical compositions for the regulation of neuronal exocytosis and thus, for the treatment of muscle spasticity and reduce and/or eliminate the asymmetry of the face and/or facial wrinkles, especially expression lines.

Wrinkles are wrinkles that appear as a result of tension of facial muscles responsible for facial expressions on the face. Wrinkles are usually on the forehead, about the transto between the eyebrows, around the mouth and/or eye. Depending on the face shape, frequency shift expressions and the presence of tics (repetitive convulsive movement, caused by involuntary contraction of one or more muscles, in this case facial muscles), wrinkles may occur even during puberty. External factors such as sun exposure, increase their depth and expression.

The botulinum toxins are widely used for reducing and/or eliminating facial wrinkles, especially serotype A (cosmetic product BOTOX®, Allergan Inc.) [Canvthers J.D. and Carruthers J.A. (1992) "Treatment of glabellar frown lines with C. botulinum-A exotoxin" J. Dermatol. Surg. Oncol. 18, IT-21; Mendez-Eastman S.K. (2003) "Botox: a review" Plast. Surg. Nurs. 23, 64-69J. Therapeutic and cosmetic treatment with BOTOX®consists of localized injections of diluted pharmaceuticals (complex of botulinum toxin type a - hemagglutinin, 500 kDa) in areas where localized muscle tension. Paralytic effects of the toxin are reversible with an average duration of 6 months [Jankovic J. and VGP F.. (1991) "Therapeutic uses of botulinum toxin" New Engl. J. Med. 324, 1186-1194; Jankovic J. (1994) "Botulmum toxin in movement disorders" Curr. Opm. Neurol. 6, 358-366]. Treatment therefore requires repeated injections of botulinum toxin. The main problem with this treatment is that you can start an immune reaction against the pharmaceutical what about the drug due to the fact, his molecules can be recognized by the immune system of the patient. The appearance of antibodies against botulinum toxin is a serious problem, because it significantly reduces the effectiveness of treatment [Jankovic J. and Brin F.M. (1991) "Therapeutic uses of botulinum toxin" New Engl. J. Med. 324, 1186-1194; Jankovic J. (1994) "Botulinum toxin in movement disorders" Curr. Opin. Neurol. 6, 358-366; Jankovic J. and Brin M.F. (1997) "Botulinum toxin: historical perspective and potential new indications" Muscle Nerve Suppl. 6, S129-S145; Davis LE (1993) "Botulinum toxin-from poison to medicine" West J. Med. 128, 25-28; Hughes A.J. (1994) "Botulinum toxin in clinical practise" Drugs 48, 888-893; 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; Borodic G.E. and Pearces L.B. (1994), "New concepts in botulinum toxin therapy" Drug Safety 11, 145-152; 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]. This loss of effectiveness of treatment with BOTOX®entails increasing the concentration of the drug in subsequent treatments, which, in turn, causes the amplification of the immune response. Alternatively, treatment of botulinum toxin serotype And discusses the application of other serotypes of botulinum toxin, such as BoTox, F BoTox and BoTox E. However, the use of pharmaceutical drugs with other serotypes cannot be considered a solution to the problem, because sooner or later, what about the immune response may reappear. In addition, treatment of botulinum toxins is expensive, mainly because of the lability and instability containing pharmaceutical preparations.

Therefore there is an urgent need to develop molecules that mimic the paralytic effects of botulinum toxins, but with a simpler and more stable molecular structure, which does not induce an immune reaction, and the cost of obtaining them is profitable. Molecule peptide meet these requirements.

At the molecular level botulinum toxins represent a protease that destroys neural proteins, which are involved in the mechanism activated by calcium ions exocytosis [Schiavo G., Rossetto O. and Montecucco C. (1996) "Bases Moleculares del tetanos at del botulismo" Investigacion y Ciencia 234, 46-55; Montecucco C. and Schiavo, G. (1994) "Mechanism of action of tetanus and botulinum neurotoxins," Mol. Environ. 13, 1-8; Schiavo G., Rosetto A., 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, 65-75J. For example, botulinum toxin And, most frequently used in clinical practice and cosmetics in terms of its application to correct facial wrinkles and asymmetry of the face and to reduce the symptomology of bronchial diseases, and reduces neuronal protein SNAP-25. This protein SNAP-25 plays an important role in neurosecretion, as he is involved in the formation the of the protein complex (known as the SNARE or complex merge), directing and controlling the release of acetylcholine, accumulated in the vesicles. The core of the specified merged complex formed by syntaxin and proteins SNAP-25, located in the presynaptic plasma membrane, and synaptobrevin or protein VAMP, localized in the plasma membrane vesicles [N. Calakos and Scheller, R.H. (1996) "Synaptic vesicle biogenesis, docking and fusion: a molecular description" Physiol. Rev. 76, 1-29; R.B. Sutton, D. Fasshauer, R. Jahn and Brunger, A.T. (1998) "Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4Å resolution" Nature 395, 347-3537. The main function of the complex merger is moving vesicles loaded with neurotransmitter (acetylcholine)to the presynaptic plasma membrane and bringing it into contact with it [N. Calakos and Scheller, R.H. (1996) "Synaptic vesicle biogenesis, docking and fusion: a molecular description" Physiol. Rev. 76, 1-29; R.B. Sutton, D. Fasshauer, R. Jahn and Brunger, A.T. (1998) "Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4Å resolution" Nature 395, 347-353]. In response to increased concentrations of calcium, there is a merging of both plasma membrane, causing the release of neurotransmitter. The specified fusion of vesicles and saarialho SNARE protein complex, thus, is the main target for control of neurosecretion. Truncation of any of the proteins forming the complex mergers, prevents its Assembly and thereby inhibits vesicular release and regulates neuronal exocytosis.

Similarly, were also described synthetic peptides obtained in accordance with the rational design or by analysis of synthetic chemical libraries, which can influence the formation of the SNARE complex by inhibiting neuronal exocytosis [Blanes-Mira C., Pastor, M. T., Valera, E., Fernández-Ballester G., J.M. Merino, L.M. Gutierrez, Perez-Paya E. and Ferrer-Montiel, A. (2003) "Identification of SNARE complex modulators that inhibit exocytosis form an α-helix-constrained combinatorial library" Biochem J. 375, 159-166].

Industrial application of this type of compounds has been limited. In the cosmetic industry have been considerable efforts to develop compounds that mimic the effects of botulinum toxins, with exceptional use in the treatment and prevention of the formation of facial wrinkles [Blanes-Mira C., Clemente J., Jodas G., Gil, A., Fernández-Ballester G., C. Ponsati, L.M. Gutierrez, Perez-Paya E. and Ferrer-Montiel, A. (2002) "A synthetic hexapeptide (Argireline®with anti-wrinkle activity" Int. J. Cosmetic Res. 24, 303-310]. More specifically, in the patent EP 1,180,524 Lipotec, S.A., describes peptides derived from the amino-terminal fragment of the protein SNAP-25, with action against wrinkles, and in international patent application WO 97/34620 also describes peptides derived from the amino acid sequence of the protein SNAP-25, specifically from carboxykinase area, or of synaptobrevin, or syntaxin, which can inhibit neuronal exocytosis.

None of the patents, sannich above, does not apply to irreversibly chemically modified derivatives of the protein SNAP-25 as a control agents neuronal exocytosis. Patent EP 1,180,524 describes a potentially reversible chemical modification of peptides aminoanisole fragment of the protein SNAP-25 for the purpose of increasing its bioavailability and ease of penetration through the blood-brain barrier and epithelial tissue, such as etherification of the side chains of aspartic and glutamic residues, which will be subsequently destroyed in vivo by intracellular esterases to release unmodified peptide responsible for the biological activity. Suddenly, the author of the present invention have found that chemically irreversible modification of the amino - and carboxyl ends of these peptides not only give them greater resistance to destruction by intracellular proteases, thereby ensuring a greater duration of their action as regulators of neuronal exocytosis, but, surprisingly, they can also increase their effectiveness in vitro 2-30 times relative efficacy of the corresponding unmodified peptide.

Modification of proteins lipid chains is described as an irreversible modification, when she performed on the amino groups present in their sequences, either by their aminobenzo or in the side chain is x lysine residues, and is reversible when she performed on tylnej groups of cysteine residues, such as a modified peptide or protein hydrolyzed in vivo relevant thioesterase [Madej, A.I. (1990) "Lipid modification of proteins and its relevance to protein targeting," J. Cell Sci. 97, 581-584; Mumby S.M. (1997) "Reversible palmitoylation of signaling proteins" Curr. Opin. Cell il. 9, 148-154]. In the prior art described examples of irreversible modifications of peptides derived fatty acid chains with the aim of increasing their effectiveness in vivo by enhancing their penetration through the skin [Lintner K. and Peschard O. (2000) "Biologically active peptides: from a laboratory bench curiosity to a functional skin care product" Int. J. Cosmet. Sci. 22, 207-218] or best immunological response to their development as potential vaccines [Gahery H., J. Choppin, Bourgault I., Fischer E., Maillere and B. Guillet J.G. (2005) "HIV preventive vaccine research at the ANRS: the lipopeptide vaccine approach," Therapie 60, 243-248], and in order to induce a greater cytotoxic effect against bacteria [B.I. Eisenstein (2004) "Lipopeptides, focusing on daptomycin, for the treatment of Gram-positive infections" Expert Opin. Investig. Drugs 13, 1159-1169] or fungi [Avrahami D. and Shai Y. (2004) "A New Group of Antifungal and Antibacterial Lipopeptides Derived from Non-membrane Active Peptides Conjugated to Palmitic Acid" J. Biol. Chem. 279, 12277-12285]. This type of modification does not always lead to efficiency in vitro of these peptides; for example, palmitoylation Tripeptide GHK (glycyl-L-histidyl-L-lysine) does not change its ability to induce collagen synthesis in f is broblast [Lintner K. and Peschard O. (2000) "Biologically active peptides: from a laboratory bench curiosity to a functional skin care product" Int. J. Cosmet. Sci. 22, 207-218]thus, a specialist in the art at the time of creation of the present invention could not predict, will increase, decrease or leave unchanged the efficiency of in vitro modification of the peptide group of the hydrocarbon compared with the corresponding unmodified peptide.

Examples exist in the prior art, describe modifications of peptides and proteins to improve their pharmacological properties, distribution, and elimination and, thus, improve their in vivo biological activity without altering their biological activity in vitro, but not in any way suggest that the potential Paglierani can increase the biological activity of the protein in vitro, on the contrary, the described examples, such as in the case of Paglialunga interferon, where activity in vitro is reduced in comparison with the activity of native interferon [Rajender Reddy K., Modi M.W. and Redder S. (1992) "Use of peginterferon alfa-2a (40 KD) (Pegasys) for the treatment of hepatitis C" Adv. Drug Deliv. Rev. 54(4), 571-86].

Unexpectedly, the present invention shows that irreversible chemical modification of peptide sequences obtained from the protein SNAP-25, can increase the efficiency of these sequences in relation to inhibition of neural e is socilize. There is no indication in the prior art that these modifications should increase the inhibitory effect of these peptides, therefore, the expert could not make a conclusion about the nature of the required modifications of the peptides to increase their ability to inhibit neuronal exocytosis.

The present invention thus represents a new solution needs, including opening irreversibly chemically modified peptide sequences obtained from the protein SNAP-25, which can inhibit neuronal exocytosis more effective and lasting way than the corresponding unmodified peptides, which are already known in the prior art.

Description of the INVENTION

The present invention represents a simple, effective and reliable solution for the regulation of neuronal exocytosis, including use in the body of a mammal a composition containing at least one peptide having the amino acid sequence derived from the amino acid sequence of the protein SNAP-25, and which irreversibly chemically modified at its amino - and/or carboxyl ends.

Thus, the first aspect of the invention relates to the peptide, which may regulate neuronal exocytosis, according to the General formula (I):

R1-AA-R2

(I)

its stereoisomers and racemic or deracemization mixtures, and its cosmetically or pharmaceutically acceptable salts, where

AA is a sequence 3-40 neighbouring

amino acids contained in the amino acid sequence of SEQ ID No.1;

R1selected from the group consisting of H or alkyl, aryl, aranceles or acyl group;

and R2selected from the group consisting of amino, hydroxyl or thiol, unsubstituted or substituted aliphatic or cyclic groups;

provided that when R1represents H or acetyl, then R2does not represent unsubstituted amino, hydroxyl or thiol.

The preferred structures of the peptides shown in the General formula (I)are structures in which

R1represents H or acetyl, or a saturated or unsaturated, linear, branched or cyclic With3-C24acyl group or polietilenglikolya polymer;

R2represents amino or hydroxyl, possibly substituted saturated or unsaturated, linear, branched or cyclic C1-C24aliphatic group;

provided that when R1represents H or acetyl, R2is not unsubstituted amino or hydroxyl.

The preferred structure is urami are patterns, in which polietilenglikolya polymer is a

where n can vary from 1 to 100, and more preferably it can vary between 1 and 5.

Additionally, preferred are structures in which R1represents an acyl group of the formula CH3-(CH2)m-CO-, where m can vary between 1 and 22.

The peptides of the present invention can exist as stereoisomers or mixtures of stereoisomers; for example, they form amino acids can have L-, D-configuration, or may be racemic independent from each other. Therefore, it is possible to obtain a mixture of isomers and the racemates or mixtures of diastereomers or pure diastereoisomers or enantiomers, depending on the number of asymmetric carbon atoms, which are isomers or mixtures of isomers. The preferred structure of the peptides according to the invention are pure isomers, i.e. enantiomers or diastereoisomers.

In the context of the present invention, the term "aliphatic group" refers to saturated or unsaturated, linear or cyclic group.

The term "hydrocarbon group" used in the present invention include, for example, alkyl, alkeline or alkyline group.

The term "alkyl group" refers to n is sydeney, linear or branched hydrocarbon group including, for example, methyl, ethyl, isopropyl, isobutyl, mpem-butyl, heptyl, dodecyl, hexadecyl, octadecyl, amyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexan etc..

The term "Alchemilla group" refers to an unsaturated, linear or branched hydrocarbon group with one or more carbon-carbon double bonds, such as vinyl group.

The term "Alchemilla group" refers to an unsaturated, linear or branched hydrocarbon group with one or more carbon-carbon triple bonds.

The term "cyclic group" refers to a closed hydrocarbon ring, which may be classified as an alicyclic, aromatic or heterocyclic group.

The term "alicyclic group" refers to a cyclic hydrocarbon group with properties similar to aliphatic groups.

The term "aromatic group" or "aryl group" refers to a mono - or polycyclic aromatic hydrocarbon group.

The term "heterocyclic group" refers to a closed hydrocarbon ring in which one or more ring atoms is an element other than carbon (e.g., nitrogen, oxygen, sulfur and so on).

The term "polietilenglikolya polymer" refers to a substituted or unsubstituted angle is Ogorodnik circuits containing the repeating unit group-CH2CH2O-.

As is understood in this technical field, the existence of high degree of substitution is not only acceptable, but recommended. Therefore, the peptides of the present invention may contain substitution. To simplify the present description, the terms "group" and "block" will be used to differentiate between chemical groups that allow for substitution or which may be substituted (the"group"), and those who do not admit the possibility of substitution, or which cannot be substituted ("block"). Thus, when the term "group" is used to describe a chemical substituent, the described chemical material includes the unsubstituted group and a group containing the atoms O, N or S.

On the other hand, when the term "block" is used to describe a chemical compound or substituent, it can only be enabled unsubstituted chemical material. For example, the expression "alkyl group" includes not only saturated alkyl substituents with an open circuit, such as methyl, ethyl, propyl, isobutyl and the like, but also alkyl substituents containing other substituents known in the prior art, such as hydroxyl, alkoxyl, amino, carboxyl, carboxamide, halogen atoms, cyano, nitro, Ala sulfonyl and others. Thus, "alkyl group" includes ether, halogenoalkane, alcohol, Tilney, carboxyl, amine, hydroxyalkyl, sulfoalkyl, guanidine, and other groups. On the other hand, the expression "alkyl" block contains only saturated alkyl substituents with an open circuit, such as methyl, ethyl, propyl, isobutyl, etc.

In the context of the present invention, "amino acid sequence derived from the amino acid sequence of the protein SNAP-25" means any amino acid sequence or fragment contained in the amino acid sequence of the protein SNAP-25, defined as SEQ ID No. 1, or any amino acid sequence that differs from a sequence contained in SEQ ID No. 1, a mutation, insertion, deletion or substitution of at least one amino acid or degeneracy of the genetic code, provided that it meets the peptide having the activity of a protein SNAP-25. Mutations, insertions or substitutions may be achieved by genetically encoded amino acids, or by non-coding amino acids, natural or synthetic, such as, for example, but without limiting them, citrulline, ornithine, sarcosine, desmosine, Norvaline, 4-aminobutyric acid, 2-aminobutyric acid, 2-aminoadamantane acid, 6-aminohexanoic acid, 1-is artillery, 2-nafcillin, 2-aminobenzoic acid, 4-aminobenzoic acid, 4-chlorophenylalanine, 2,3-diaminopropionic acid, 2,4-diaminopentane acid, cycloserine, carnitine, cystine, penicillamine, pyroglutamyl acid, titillans, hydroxyproline, ALLO-isoleucine, ALLO-threonine, isonicotinoyl acid, Soeren, phenylglycine, a statin, β-alanine, norleucine, N-methylaminomethyl, β-amino acid or γ-amino acids and their derivatives. The list of synthetic amino acids can be found in the article "Unusual amino acids in peptide synthesis" by D.C. Roberts and F. Vellaccio, in The Peptides, Vol.5 (1983), Chapter VI, E. Gross and J. Meienhofer, Eds, Academic Press, New York, USA, or commercial directory of companies specializing in this sector, such as, for example, NeoMPS, Bachem, Novabiochem, Sigma-Aldrich, Peptides International, Advanced ChemTech, Chem-lmpex, Maybridge Chemical, Chirotech Technology, Peninsula Laboratories or RSP Amino Acid Analogues among others.

Among the peptides of the invention derived from the amino acid sequence of SNAP-25, a particular SEQ ID No. 1 and chemically modified irreversible manner, preferred are sequences that have amino acid sequence contained in the sequence of aminobenzoic region of the protein SNAP-25, a particular SEQ ID No. 2, or carboxykinase region of the protein SNAP-25, a particular SEQ ID No. 3, more preferably contained in the region between residues 10-22, op is delannoy SEQ ID No. 4, or contained in the region between residues 25-40 defined by SEQ ID No. 5, or contained in the region between residues 65-81 defined by SEQ ID No. 6, or contained in the region between residues 181-206 defined by SEQ ID No. 7, more specifically contained in the region between residues 12-19 defined by SEQ ID No. 8, or contained in the region between residues 26-38 defined by SEQ ID No. 9, or contained in the region between residues 68-79 defined by SEQ ID No. 10 SEQ, and specifically contained in the region between residues 12-17 defined by SEQ ID No. 11.

The invention also includes peptides, which are essentially homologous irreversibly chemically modified peptides derived from the amino acid sequence of the protein SNAP-25. By "essentially homologous peptides" understand amino acid sequence that is at least 60%, preferably 80% and more preferably 95% identical to the sequence SEQ ID No. 1. "Percent identity" refers to the percentage of amino acids that are identical between two amino acid sequences are compared after optimal alignment of these sequences, where this percentage is only statistical and the differences between the two amino acid pic what egovernance randomly distributed along the sequence. Under "optimal alignment" understand the alignment of amino acid sequences, giving a higher percentage identity. The percentage identity is calculated by determining the number of identical positions where the amino acid is identical in the two compared sequences, dividing the number of identical positions by the number of compared positions and multiplying the result by 100 to obtain the percentage identity between the two sequences. Comparison of sequences between two amino acid sequences can be performed manually or by computer programs, such as the BLAST algorithm (Basic Local Alignment Search Tool), which can be accessed through the Internet on the website http://www.ncbi nim nlm.gov/BLAST/.

Cosmetically or pharmaceutically acceptable salts of the peptides, proposed in accordance with this invention are included in the scope of the present invention. The term "cosmetically or pharmaceutically acceptable salts" includes salts commonly used to obtain salts with metals or salts accession acids or salts accession of organic acids (such as, for example, acetate, citrate, oleate, triptorelin, oxalate or gluconate) or salts accession of inorganic acids (such as, for example, chloride, sulfate, borate or carbonate). Nature is not important provided it is cosmetically or pharmaceutically acceptable. Cosmetically or pharmaceutically acceptable salts of the peptides according to the invention can be obtained by conventional methods known from the prior art.

Synthesis of peptides according to the invention can be carried out by conventional means known from the prior art, such as, for example, various methods of solid-phase peptide synthesis [J.M. Stewart and J.D. Young (1984) Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois. M. Bodanzsky and A. Bodanzsky (1984) "The practice of Peptide Synthesis", Springer Verlag, New York; Lloyd-Williams, P., Albericio, F. and Giralt, E. (1997) "Chemical Approaches to the Synthesis ofPeptides and Proteins" CRC, Boca Raton (FL, USA)], synthesis in solution, the combination of solid-phase synthesis and methods of synthesis in solution or methods of enzymatic synthesis of [Kullmann W. (1980) "Proteases as catalysts for enzymic syntheses ofopioid peptides" J. Biol.Chem. 255, 8234-8238]. The peptides can also be obtained by fermentation of the bacterial strain, which is unmodified or modified by means of genetic engineering to obtain the desired sequences, or by controlled hydrolysis of proteins of animal or vegetable origin, preferably of plant origin, which releases the peptide fragments containing at least the desired sequence.

For example, a method of obtaining a peptide according to the invention is the persons, when the peptide fragment according to the invention, having a free carboxyl group or its reactive derivative, is subjected to the interaction with an additional fragment having an amino group with at least one free hydrogen atom followed by the formation of amide linkages, where the functional groups of these fragments, which do not participate in the formation of the amide bond, if present, conveniently protected with temporary or permanent protective groups.

Another example of a method of obtaining a peptide according to the invention is a method in which a peptide fragment according to the invention, containing a leaving group such as, for example, tosyl, methylsulphonyl group and halogen, is subjected to the interaction with an additional fragment having an amino group with at least one free hydrogen atom by reaction of nucleophilic substitution, where the functional groups of these fragments, which do not participate in the formation of N-S, if they are present, conveniently protected with temporary or permanent protective groups. Examples of protective groups, their introduction and removal are described in the literature [Greene, T.W. (1981) "Protective groups in organic synthesis" John Wiley & Sons, New Cork; B. Atherton and Sheppard R.C. (1989) "Solid Phase Peptide Synthesis: A practical approach," IRL Oxford University Press]. The term "protective GRU is p ' also includes a polymeric substrate, used in solid-phase synthesis.

The peptides according to the invention can be administered to regulate neuronal exocytosis in any way, ensure the contact of the compounds with the site of its action in the body of a mammal, preferably a human, in the form of, compositions containing them. In this sense, the invention provides a cosmetic or pharmaceutical composition comprising at least one peptide of General formula (I) or its cosmetically or pharmaceutically acceptable salts. These compositions can be obtained by conventional methods known to experts in this field of technology.

The peptides according to the invention are used in cosmetic or pharmaceutical compositions according to the present invention in a cosmetically or pharmaceutically effective concentration to achieve the desired effect; preferably between 0,00000001% (by weight) and 20% (by weight); preferably between 0,00001% (by weight) and 10% (by weight) and more specifically between 0,0001% (by weight) and 5% (by weight).

The peptides of the present invention have different solubility in water, depending on the nature of their sequence, or their modifications, by amino - and carboxyterminal ends. Peptides, which are not soluble in water, can be solubilisation in about ycnih cosmetically or pharmaceutically acceptable solvents, such as, for example, ethanol, propanol or isopropanol, propylene glycol, glycerin, butyleneglycol or polyethylene glycol.

Pharmaceutically effective amount of the peptides and/or pharmaceutical compositions according to the invention, which should be introduced for the treatment of pathological States, as well as their dosage will depend on several factors, including age, condition of the patient, the severity of the disorders or diseases, manner, and frequency of injection of specific peptides that will be used.

Pharmaceutical compositions containing the peptides according to the invention can have any shape for insertion, for example, solid or liquid, and can be introduced by any suitable means, such as oral, intranasal, parenteral, rectal, topical or transdermal what they will include the necessary pharmaceutical excipients for the manufacture of the desired form of administration. In the context of the present invention, the term "parenterally" includes subcutaneous, dermal, intravascular injection, such as intravenous, intramuscular, intracerebral channel, intracranial, intra-articular, vnutriobolochechnoe and intraperitoneally injections, as well as any other similar methods of injection or infusion. An overview of the different pharmaceutical forms of administration of medicinal PR the products and the necessary excipients for their manufacture may be found, for example, in the "Tratado de Farmacia Galenica", C. Fault i Trillo, 1993, Luzan 5, S.A. Ediciones, Madrid.

The peptides according to the invention can also be incorporated into cosmetic or pharmaceutical systems with delayed release and/or system of carriers, such as liposomes, milecastle, microparticles and nanoparticles, sponges, vesicles, micelles, military, microspheres and nanospheres, liposphere, millicapsules, microcapsules and nanocapsules, as well as in micro-emulsions and nano-emulsion, in order to achieve better penetration of the active ingredient and/or improve its pharmacokinetic and pharmacodynamic properties. Drugs controlled release can be obtained by methods known in the prior art, and can be introduced through, for example, local administration, including adhesive patches, or oral, rectal or subcutaneous implantation, or by direct implantation in a specific part of the body, and should preferably release a relatively constant number of peptides according to the invention. The number of peptide contained in the drug controlled release, will depend on, for example, site of administration, the kinetics and duration of release of the peptide according to the invention, as well as from the nature of the state that treat or prevent.

The peptides of the present invention can also be adsorbed on the firmness of the amounts of organic polymeric or mineral substrates, such as, for example, talc, bentonite, silica, starch or maltodextrin.

Cosmetic or pharmaceutical preparations containing peptides of the present invention can be used in various types of drugs for local application, such as, for example, without limiting them, creams, emulsions of oil and/or silicone in water, emulsion water in oil and/or silicone oils, lotions, balms, foams lotions, gels, liniments, sera, Soaps, creams, foam, ointment, mousse, fondant, plates, pencils and sprays, including "fixed" and "rinse-off" products, and can also be incorporated by techniques known to experts in the art, various types of solid substrates, such as woven materials, hydrogels, adhesive (or non-adhesive) patches or face masks, or can be incorporated into various cosmetic products, such as cosmetics, lotions, cleansing milk to remove make-up, creams, eye shadow and lipstick.

The peptides may also be included in the materials for the manufacture of coatings, which are in direct contact with the skin of the body, so that the peptides according to the invention are released by biodegradation system with fixation on the fabric or by the friction of the cover with the body, under the action of moisture, body, skin pH or body temperature. P is emery coatings, materials and tools for immobilization of peptides in tissues, including microencapsulation described in the literature and known from the prior art [Schaab C.K. (1986) "Impregnating Fabrics With Microcapsules", HAPPI May 1986; Nelson G. (2002) "Application of microencapsutation in textiles" Int. J. Pharm. 242, 55-62]. Preferred coatings are bandages.

Cosmetic or pharmaceutical composition of the present invention can be applied in areas of the body requiring treatment or care by subcutaneous injection, intradermal injection, steam wraps or by iontophoresis in order to achieve better penetration of the active ingredient. The scope is determined by the nature of the state, which should be treated. The preferred area of application is the area of the forehead with wrinkles, as well as the space between the eyebrows, wrinkles and fine lines around the mouth and/or eye.

Cosmetic or pharmaceutical composition, as claimed in the present invention may contain additional ingredients commonly used in compositions to treat, cleanse and treat the skin, such as, for example, without limitation, emulsifying agents, softeners, organic solvents, conditioners for skin care, such as, for example, moisturizers, alpha-hydroxyacids, moisturizers, vitamins, pigmen is s or dyes gelling polymers, thickeners, plasticizers, agents, anti-wrinkle agents, which can reduce or cure under-eye bags, whitening or depigmenting agents, exfoliating agents, anti-ageing agents, agents that traps free radicals and/or atmospheric pollution, agents that inhibit NO synthase, antioxidants, components against glycation, antimicrobial agents, antifungal agents, agents stimulating the synthesis of dermal or epidermal macromolecules and/or capable of inhibiting their degradation, such as, for example, agents stimulating collagen synthesis, agents stimulating the synthesis of elastin, the agents stimulating synthesis decorin, agents stimulating the synthesis of laminin, agents that inhibit the degradation of collagen, agents that inhibit the degradation of elastin, the agents which stimulate the proliferation of fibroblasts, agents stimulating the proliferation of keratinocytes, agents stimulating the differentiation of keratinocytes, agents stimulating the synthesis of lipids and components of the stratum corneum (ceramides, fatty acids and so on), relaxing the skin agents, agents stimulating the synthesis of glycosaminoglycans and collagen, the substance that gives the density, agents against stretch marks, soothing agents, anti-inflammatory agents, agents acting on capillary circulation and/and and microcirculation, agents acting on cell metabolism, agents stimulating and/or inhibiting the synthesis of melanin, agents to improve dermal-epidermal connections, preservatives, perfumes, chelating agents, vegetable extracts, essential oils, extracts of seafood, agents, resulting biofermentation, mineral salts, cell extracts and sunscreens agents (organic or mineral photoprotective agents that are active against ultraviolet a and b rays), provided that they are physically and chemically compatible with other components of the composition and especially with the peptides of General formula (I)contained in the composition of the present invention. The nature of these additional ingredients can be synthetic or natural, such as, for example, plant extracts, or they may be the result of the process of biofermentation.

An additional aspect of the present invention relates to cosmetic or pharmaceutical composition containing a cosmetically or pharmaceutically effective amount of at least one peptide according to the invention, and also a cosmetically or pharmaceutically effective amount of at least one extract with activity against wrinkles and/or aging, such as, for example, without limitation to them is, extracts of Vitis vinifera (grapes), Rosa canina (rose hips), Curcuma longa (turmeric), Iris pallida (iris pale), Theobroma cacao (cocoa), Ginkgo biloba (gingko biloba) or Dunaliella salina (Dunaliella), or also at least one synthetic compound, extract or product biofermentation with activity against wrinkles and/or aging, such as, for example, without limiting them, Matrixyl®manufactured by Sederma, Vialox®or Syn-ake®manufactured by Pentapharm, Myoxinol™manufactured by Cognis, Algisum With®or Hydroxyprolisilane CN®produced by Exsymol, Argireline®, LeuphasyI®, Aldenine®, Decorinyl®, Decorinol®or Lipochroman®manufactured by Lipotec, Kollaren®produced by the Institut Europeen de Biologie Cellulaire, Collaxyl®or Quintescine®produced by Vincience, antagonists of CA2+channel, such as alverine, salts of manganese or magnesium, certain secondary or tertiary amines, vitamin a and its derivatives, idebenone and its derivatives, coenzyme Q10 and its derivatives, Boswellia acid and its derivatives, gamma-aminobutyric acid or agonists chloride channel.

The compositions of the present invention may contain or may be introduced together with analgesic compounds and/or anti-inflammatory compounds to reduce the swelling and irritation associated with sensitive skin. Connection type steroids, such to the to hydrocortisone, connection nonsteroidal type, such as paracetamol or acetylsalicylic acid, or natural extracts or essential oils with intrinsic analgesic and anti-inflammatory activity.

The peptides according to the invention possess a mechanism of action similar to the mechanism of action of botulinum toxin, inhibiting neuronal exocytosis, therefore, we can assume that the peptides will be effective for the treatment of facial wrinkles and/or asymmetry of the face, and to treat conditions that represent muscle spasticity, such as dystonia, strabismus, blepharospasm, torticollis, ticks, etc.

An additional aspect of the present invention thus relates to the use of at least one peptide of General formula (I) in the manufacture of cosmetic or pharmaceutical compositions for treating conditions mammals, preferably humans, which require regulation of neuronal exocytosis. Another aspect of the present invention relates to the use of at least one peptide of General formula (I) for obtaining a cosmetic or pharmaceutical composition for the treatment, purification or skin care. An additional aspect of the present invention relates to the use of at least one irreversibly chemically modified peptide derived from the amino acid effects the successive protein SNAP-25, for the production of cosmetic or pharmaceutical compositions for reducing and/or eliminating asymmetry and facial wrinkles, preferably facial wrinkles. Another additional aspect of the present invention relates to the use of at least one irreversibly chemically modified peptide derived from the amino acid sequence of the protein SNAP-25, for the production of cosmetic or pharmaceutical compositions for the treatment of neurological disorders or pathologies, representing muscle spasticity, such as dystonia, strabismus, blepharospasm, torticollis, ticks, etc.

Another aspect of the present invention relates to a cosmetic or pharmaceutical method for the treatment of those conditions mammals that require regulation of neuronal exocytosis, preferably humans, comprising introducing an effective amount of at least one peptide of General formula (I), preferably in the form containing cosmetic or pharmaceutical compositions. The present invention additionally relates to a cosmetic or pharmaceutical method of reducing and/or eliminating facial wrinkles or treatment of facial asymmetry, comprising applying to the skin a cosmetic or pharmaceutical composition containing at least one peptide from which briteney or its cosmetically or pharmaceutically acceptable salts. The present invention also relates to a cosmetic or pharmaceutical method for the treatment of neurological disorders or pathologies associated with muscle spasticity, such as dystonia, strabismus, blepharospasm, torticollis, ticks, etc. comprising applying a cosmetic or pharmaceutical composition containing at least one peptide according to the invention or its cosmetically or pharmaceutically acceptable salts.

Frequency of application can vary widely depending on the needs of each subject, the application period is from once per month to 10 times per day, preferably from once per week up to 4 times per day, more preferably from three times per week to two times daily, more preferably once a day.

Preferred cosmetic or pharmaceutical method is a method in which the application is carried out on those areas of the face or forehead, where there are wrinkles, preferably on the wrinkles around the mouth and/or eyes, and/or wrinkles on the forehead and/or wrinkles in the space between the eyebrows.

DESCRIPTION of GRAPHIC MATERIALS

Figure 1 shows that in the presence of the peptides according to the invention in a concentration of 0.1 mm, the release of [3H]-L-glutamate from primary cultures of hippocampal neurons in rats was ingibirovalo more than 40%of that evidence is the duty to regulate about these compounds are inhibitors of neuronal exocytosis. Inhibition of release of [3H]-L-glutamate unmodified peptide derived from the amino acid sequence of the protein SNAP-25 defined in SEQ ID No. 11, in the same concentration was only 5%, while the concentration in approximately 30 times larger (3 mm) was required to achieve the levels of inhibition, which may be comparable with those for irreversibly modified peptides.

EMBODIMENTS of the INVENTION

The following provides specific examples are useful to illustrate the nature of the present invention. These examples are included merely for illustration and should not be interpreted as limitations of the claimed invention.

General methodology

Chemical synthesis

All procedures of synthesis performed in polypropylene syringes equipped with porous polyethylene discs. All reagents and solvents are as for the synthesis and used without any additional processing. Solvents and reagents are removed by suction. Removal of the Fmoc group perform in a mixture of popperian-DMF (dimethylformamide) (2:8, vol/about.) (1×1 min, 1×5 minutes; 5 ml/g resin) [Lloyd-Williams P., Albericio F. and Giralt, E. (1997) "Chemical Approaches to the Synthesis of Peptides and Proteins" CRC, Boca Raton (FL, USA)]. Washing between stages of removal protection, combination and even the AZ removal protection was performed using DMF (3×1 min), using 10 ml solvent/g resin. Of combination reaction was performed with 3 ml of solvent/g of resin. Control combinations performed by ninhydrin test [Kaiser, E., Colescott R.L, Bossinger C.D. and Cook P.I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides" Anal. Biochem. 34, 595-598J. All synthetic transformations and washing were performed at 25°C.

Mass spectroscopic analysis elektrorazpredelenie performed in the device LCMS-QP 8000 Shimadzu (Kyoto, Japan)using a mixture of MeCN:H2O 4:1 (+0,1% TFA) as mobile phase and at a flow rate of 0.2 ml/min

Reduction

Abbreviations used for amino acids, consistent with the Commission rules IUPAC-IUB biochemical nomenclature presented in EIG. J. Biochem. (1984) 138, 9-37 and J. il. Chem. (1989) 264, 633-673.

AM, 2-[4-aminomethyl-(2,4-acid)]-venexiana acid; BoNT, botulinum toxin serotype A; cps, CP; DCM, dichloromethane; DIEA, N,N'-diisopropylethylamine; DIPCDI, N,N'-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; DPPC, dipalmitoylphosphatidylcholine; EQ., equivalent; Fmoc, fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; INCI, international Nomenclature of Cosmetic ingredients; MUNA, p-methylbenzhydrylamine; MeCN, acetonitrile; MLV, multi-layered vesicles; Meon, methanol; NMP, N-organic; Pbf, 2,2,4,6,7-pentamethylcyclopentadiene-5-sulfonyl; PEG, polyethylene glycol; PEGn, -[NH-CH2-(CH2 CH2O)3-(CH2)3-NH-CO-CH2CH2-CO-]n;.rpm, revolutions per minute; SNAP-25 is associated with the uptake protein (25 kDa); tBu, tert-butyl; TFA, triperoxonane acid; THF, tetrahydrofuran; ULV, monolayer vesicles.

EXAMPLE 1

Obtaining Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-AM-MBHA

151, 3mm g resin Fmoc-AM-MBHA with functionalization 0,628 mmol/g (95 mmol, 1 EQ.) was treated with a mixture of piperidine-DMF according to the described General Protocol in order to remove the Fmoc group. 154,1 g Fmoc-L-Arg(Pbf)-OH (237 mmol, 2.5 EQ.) included in the unprotected resin in the presence of DIPCDI (36,6 ml, 237 mmol, 2.5 EQ) and HOBt (35,6 g, 237 mmol, 2.5 EQ.) using DMF as solvent for 1 hour.

The resin was then washed as described in the General methods and processing for removal of the protective Fmoc group was repeated to include the following amino acid. Following described protocols, 154,1 g Fmoc-L-Arg(Pbf)-OH (237 mmol, 2.5 equiv.) of 87.5 g of Fmoc-Gln-OH (474 mmol, 5 EQ), 88,2 g Fmoc-L-Met-OH (237 mmol, 2.5 equiv.) to 105.3 g of Fmoc-L-Glu(OtBu)-OH (237 mmol, 2.5 EQ.) and to 105.3 g of Fmoc-L-Glu(OtBu)-OH (237 mmol, 2.5 EQ) are successively combined in the presence of each combination of 36.5 g of HOBt (237 mmol, 2.5 EQ) and 36.6 ml DIPCDI (237 mmol, 2.5 equiv.) except for the stage include Fmoc-L-Gln-OH, in which use 71,3 g HOBt (474 mmol, 5 EQ.).

The obtained Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-AM-MVNA washed with DMF (5×1 min), DCM (4×1 min), diethyl ether (4×1 min) and su is at in a vacuum.

EXAMPLE 2

Obtaining CH3-(CH2)m-CO-Glu-Glu-Met-Gln-Arg-Arg-NH2

Aminobenzene the Fmoc group in the party 1.68 g of Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-AM-MBHA (0.5 mmol, 0,296 mmol/g, 1 EQ.) delete, as described in the General methods, and CH3-(CH2)mCOOH (5 mmol, 10 EQ), pre-dissolved in DMF (10 ml), include the presence of 770 mg of HOBt (5 mmol, 10 EQ.) and 770 μl of DIPCDI (5 mmol, 10 EQ). The interaction was allowed to proceed for 15 hours, after which the resin washed with THF (5×1 min), DCM (5×1 min), DMF (5×1 min), Meon (5×1 min), DMF (5×1 min), THF (5×1 min), DMF (5×1 min), DCM (4×1 min), ether (3×1 min) and dried in vacuum.

1,00 g dry peptidyl resin is treated with 15 ml of TFA-iPr3Si-H2O (90:5.5) for 2 hours at room temperature. The filtrate is collected in a cold diethyl ether (100 ml), centrifuged for 5 minutes at 4000 rpm and the ethereal solution is decanted. Rinse repeat with ether 5 times. The final precipitate is dried in vacuum.

mthe received quantitypuritytheoretical MM (mol. weight)experimental MM (mol. weight)
6291,2 mg 88,2%[M+H]+=972,5[M+H+]=973,8
[M+2H+/2]=487,5
8240,1 mg87,2%[M+H]+=1000,5[M+H+]=1001,8
[M+2H+/2]=501,5
12327,5 mg80,7%[M+H]+=1056,6[M+H+]=1057,8
[M+2H+/2]=529,5
14292,8 mg80,9%[M+H]+=1084,6[M+H+]=1085,9
[M+2H+/2]=543,7
20 233,7 mg85,1%[M+H]+=1168,74(M+N+]=1170,0
[M+2H+/2]=585,7

EXAMPLE 3

Obtaining Ac-PEGn-Glu-Glu-Met-Gln-Arg-Arg-NH2

Aminobenzene the Fmoc group of the party 321,0 mg Fmoc-Glu(OtBu)-Glu(OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-AM-MBHA (0,095 mmol, 0,296 mmol/r, 1 EQ.) delete, as described in the General methods, and Fmoc-PEG1-OH (2.5 EQ.), pre-dissolved in NMP, add in the presence of 36.5 mg HOBt (0,237 mmol, 2.5 EQ.) and 36.6 ál of DIPCDI (0,237 mmol, 2.5 EQ.) within 40-60 minutes. Remove aminobenzene the Fmoc group, as described in the General methods, and reactions introduction of Fmoc-PEG1-OH and Fmoc removal perform (n-1) times, where n=1-100, to obtain various derivatives. Acetylation of aminobenzoic region performed using Ac2O (2.5 EQ.) and DIEA (2.5 EQ) in DMF for 30 minutes

Resin Ac-PEGn-Glu(OtBu)-Glu(OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-AM-MBHA washed with DMF (5×1 min), DCM (4×1 min), diethyl ether (4×1 min) and dried in vacuum.

of 22.4 mg of Ac-PEGn-Glu(OtBu)-Glu(OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-AM-MBHA process of 1.57 ml of a mixture of TFA-iPr3Si-H2O (95.2,5 and 2.5) for 5 minutes at 0°C., then 90 minutes at room temperature. The filtrate is collected in a cold diethyl ether (10 ml),centrifuged for 5 minutes at 4000 rpm and the ethereal solution is decanted. Rinse repeat ether 5 times. The final oily residue re-dissolved in a mixture of MeCN:H2O 1:1 and lyophilizers.

nthe received quantitypurityTheoretical MMExperimental MM
17.7 mg72%[M+H]+=1191,6[M+2H+/2]=596,4
[M+3H+/3]=398,0
210.0 mg60%[M+H]+=1494,7[M+2H+/2]=747,6
[M+3H+/3]=498,7
310,4 mg64%[M+H]+=1796,1[M+2H+/2]=898,7
[M+3H+/3]=599,6
[M+4H+/4]=449,8
413,7 mg65%[M+H]+=2099,5[[M+2H+/2]=1050
[M+3H+/3]=700,4
[M+4H+/4]=525,4
[M+5H+/5]=420,5
510,4 mg65%[M+H]+=2400,3[M+2H+/2]=1201,1
[M+3H+/3]=801,1
[M+4H+/4 601,0
[M+5H+/5]=481,0
[M+6N+/6]=400,9

EXAMPLE 4

Obtaining Ac-Glu-Glu-Met-Gln-Arg-Arg-NH-(CH2)s-CH3

2,10 g Fmoc-L-Arg(Pbf)-OH (3,23 mmol, 1 EQ.), dissolved in 20 ml of DCM, were added 500 μl of DIEA (2.9 mmol, of 0.90 EQ), include in dry 2-chlorotrityl resin (2.0 g, 3.3 mmol). Leave to mix for 5 minutes, then add 1 ml of DIEA (5.9 mmol, 1,81 EQ). Left to interact for 40 minutes. The remaining chloride group protects the processing of 1.6 ml Meon.

Aminobenzene the Fmoc group is removed as described in the General methods, or 3.24 g of Fmoc-L-Arg(Pbf)-OH (5 mmol, 5 EQ) include 1 mmol aminoaniline resin Fmoc-L-Arg(Pbf)-CITrt®in the presence of DIPCDI (770 μl, 5 mmol, 5 EQ.) and HOBt (770 g, 5 mmol, 5 EQ) using DMF as a solvent for 1 hour. The resin is then washed as described in the General methods, and processing for removal of the protective Fmoc group again, to include the following amino acid. Following the protocols of 1.84 g of Fmoc-L-Gln-HE (5 IMO is ü, 5 equiv.) 1.86 g Fmoc-L-Met-OH (5 mmol, 5 equiv.) 2,12 g Fmoc-L-Glu (OtBu)-OH and 2.12 g of Fmoc-L-Glu(OtBu)-OH (5 mmol, 5 EQ.) combine consistently in the presence of each combination of 770 mg of HOBt (5 mmol, 5 EQ) and 770 μl of DIPCDI (5 mmol, 5 equiv.) except for the stage, which include Fmoc-L-Gln-OH and add 1.54 g HOBt (10 mmol, 10 EQ).

Aminobenzene the Fmoc group is removed as described in the General methods, peptidyl resin is treated for 30 minutes at 2.36 ml of acetic anhydride (25 mmol, 25 EQ.) in the presence 4,28 ml DIEA (25 mmol, 25 EQ.) using DMF as solvent, washed with DMF (5×1 min), DCM (4×1 min), diethyl ether (4×1 min) and dried in vacuum.

Fully protected peptide [Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-OH] receiving, processing for 5 minutes peptidyl resin, previously dried in vacuum in the presence of KOH, 3%solution of TFA in DCM. The filtrate is collected in a cold diethyl ether and the process is repeated three times. The ether solution evaporated to dryness on a rotary evaporator at room temperature; the precipitate is re-suspended in 50%MeCN in H2O and lyophilizers. 279 mg of the obtained crude product (367 mmol) is weighed into a container, add 3 EQ. CH3-(CH2)s-NH2and 30 ml of anhydrous DMF. Add 120 ál of DIPCDI (2 EQ.) and left to interact with stirring on a magnetic stirrer at 47°C. the Reaction controlling the t by means of HPLC (high performance liquid chromatography) on the extinction of the original product, it ends after 24 hours. The solvent is evaporated to dryness and co evaporated twice with DCM. The precipitate [Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln-Arg(Pbf)-Arg(Pbf)-NH-(CH2)s-CH3] re-suspended in 50 ml of a mixture of TFA-iPr3Si-H2O (90:5.5) and allowed to interact for 30 minutes at room temperature. Add 250 ml of cold diethyl ether, the solvent is evaporated on a rotary evaporator, conduct two additional joint evaporation with ether. The precipitate is dissolved in a mixture of 50%MeCN in H2O and lyophilizers.

sthe received quantitypurityTheoretical MMExperimental MM
11391,2 mg92,2%[M+H]+=1058,3[M+H+]=1057,9
[M+2H+/2]=529,8
15424,5 mg90,5%[M+H]+=1114,74[M+H+]1114,0
[M+2H+/2]=557,6

EXAMPLE 5

According to the General protocols described in Examples 1-4, a routine manner by varying the nature of the reagents and the sequence of the peptide were obtained following irreversibly chemically modified peptides derived from the sequence of the protein SNAP-25, is included in the scope of the present invention.

EXAMPLE 6

Analysis activity irreversibly chemically modified peptides derived from the protein SNAP-25 in respect of neuronal exocytosis [3H]-L - glutamate

To determine inhibit whether the peptides to posovremeni neuronal exocytosis of neurotransmitters, their activity in relation to the release of L-glutamate, a neurotransmitter primary cultures of hippocampal neurons of rats were analyzed as follows. Exocytosis of the neurotransmitter in neural cultures can be obtained by electrical depolarization of the cells. Primary cultures of hippocampal nuclei of rats were obtained using conventional methods [Blanes-Mira, S., J.M. Merino, Valera, E., Fernandez-Ballester, G., Gutierrez, L.M., Viniegra, S., Perez-Paya E. and Ferrer-Montiel A. Small peptides patterned after the N-terminus domain of SNAP25 inhibit SNARE complex assembly and regulated exocytosis" J. Neurochem. 88, 124-135], and maintained in culture for 14 days in a thermostat at 37°C and 5%CO2. Cultures were incubated with [3H]-L-glutamine in order to load their [3H]-glutamate for 3 hours at 37°C. an Excess of [3H]-L-glutamine is then washed and the culture incubated with 0.1 mm of the studied peptides for 1 hour at 37°C. [3H]-L-glutamate released by depolarization 75 mm KCl and 2 mm CaCl2, buffered physiological buffer for 10 minutes at 37°C. the Culture medium is collected and the amount of [3H]-L-glutamate determine quantitatively the level meter beta radiation. The results normalize relative to the release of [3H]-L-glutamate in the absence of peptide and corrected relative to basal release in the absence of calcium.

EXAMPLE 7

Receipt the cosmetic composition, containing CH3-(CH2)14-CO-Glu-Glu-Met-Gln-Arg-Arg-CONH2

The following composition was prepared as described in the present invention.

The components of Phase a were weighed into a sufficiently large reactor and the mixture was heated at 80°C. to melt the waxes. The components of Phase b were weighed in a container that is appropriate for all content, and was heated at 70°C. Phase And slowly added to Phase b with intensive stirring, and then under stirring add Phase C. Upon completion of addition the mixture was left to cool with careful stirring and, when the mixture reached room temperature, was added an aqueous solution of CH3-(CH2)14-CO-Glu-Glu-Met-Gln-Arg-Arg-CONH2and lecithin, the mixture is homogenized and the pH was adjusted with triethanolamine, if needed.

The resulting cream has a pH between 6 and 7 and the viscosity 10000-15000 SDR (6/50).

INGREDIENT (INCI nomenclature)wt.%
PHASE AND
MINERAL OIL8,0
STEARIC ACID2,4
Cetearyl ALCOHOL1,6
BEESWAX 0,8
PHASE
GLYCERIN2,4
WATER63,4
PHASE
CARBOMER0,3
TRIETHANOLAMINE0,9
FASO
WATER15,0
CH3-(CH2)14-CO-Glu-Glu-Met-Gln-Arg-Arg-CONH2(0,05%)5,0
LECITHIN0,4

EXAMPLE 8

Obtaining liposomes containing CH3-(CH2)14-CO-ELEEMQRRADQLA-NH2Dipalmitoylphosphatidylcholine (DPPC) is weighed and dissolved in chloroform. The solvent is evaporated in vacuo to obtain a thin layer of phospholipid, and this layer hydratious, processing at 55°C. aqueous solution containing the peptide in the desired concentration (containing Phenonip®), receiving MLV liposomes (monolayer). Liposomes ULV (multilayer) receive, immersing MLV liposomes in an ultrasonic bath at 55°C for 8 cycles of 2 minutes with 5-minute intervals.

INGREDIENTwt.%
DIPALMITOYLPHOSPHATIDYLCHOLINE4,0
CH3-(CH2)14-CO-ELEEMQRRADQLA-NH20,2
PHENONIP®0,5

According to the first aspect, the present invention relates to a peptide of General formula (I):

its stereoisomers, its cosmetically and pharmaceutically acceptable salts and their mixtures, where:

AA is a sequence 3-40 adjacent amino acids contained in the amino acid sequence of SEQ ID No. 1;

R1selected from the group consisting of H or alkyl, aryl, aranceles or acyl group; and

R2selected from the group consisting of amino, hydroxyl or thiol, unsubstituted or substituted aliphatic or cyclic groups,

provided that when R1represents H or acetyl, R2is not unsubstituted amino, hydroxyl or thiol.

According to the second important aspect, the peptide of General formula (I) R1represents preferably a saturated or unsaturated, linear, branched or cyclic With3-C24acyl. R1present is employed, a preferred acyl of the formula CH 3-(CH2)m-CO-, where m can vary between 1 and 22.

According to another important aspect of the invention, the peptide of General formula (I) R1represents preferably polietilenglikolya polymer.

According to another important aspect of the invention, the peptide of General formula (I) R1represents preferably polietilenglikolya polymer with a molecular weight of from 200 to 35,000 daltons.

According to another important aspect of the invention, the peptide of General formula (I) R1represents preferably polietilenglikolya polymer of the formula

where n can vary between 1 and 100. In a preferred aspect of the invention, n can vary between 1 and 5.

According to an important aspect of the invention, the peptide of General formula (I) R2represents preferably amino or hydroxyl, unsubstituted or substituted saturated or unsaturated, linear, branched or cyclic With1-C24aliphatic groups.

According to an important aspect of the invention, the peptide of General formula (I) AA preferably consists of a sequence 3-40 adjacent amino acids contained in the sequence selected from the group consisting of amino acid sequences MAEDADMRNELEEMQRRADQL, ADESLESTRRMLQLVEESKDAGI, ELEEMQRRADQLA, ELEEMQRRADQL, ELEEMQRRADQ, ELEEMQRRAD, ELEEMQRA, ELEEMQRR, LEEMQRRADQL, LEEMQRRADQ, LEEMQRRAD, LEEMQRRA, LEEMQRR, EEMQRRADQL, EEMQRRADQ, EEMQRRAD, EEMQRRA, EEMQRR, LESTRRMLQLVEE, NKDMKEAEKNLT, KNLTDL, IMEKADSNKTRIDEANQRATKMLGSG, SNKTRIDEANQRATKMLGSG, TRIDEANQRATKMLGSG, DEANQRATKMLGSG, NQRATKMLGSG and QRATKMLGSG.

According to another important aspect, the present invention relates to a method for producing a peptide of General formula (I), which is based on solid-phase peptide synthesis.

According to another important aspect, the present invention relates to a method for producing a peptide of General formula (I), in which the use of a protective group selected from the group consisting of Fmoc/mpem-butyl, Fmoc/trail and Fmoc/allyl.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical composition containing a cosmetically or pharmaceutically effective amount of at least one peptide of formula (I) and at least one cosmetically or pharmaceutically acceptable excipient or adjuvant.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions containing at least one peptide of General formula (I), included in a cosmetically or pharmaceutically acceptable system and/or a slow release carrier selected from the group consisting of liposomes, millicapsules, microcapsules and nanocapsules, sponges, vesicles, micelles, mallister, microspheres, nanoparticles, lipofen, microemu the sea, nano-emulsions, milicast, microparticles and nanoparticles.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions containing at least one peptide of General formula (I)adsorbed on solid organic polymer or inorganic substrate selected from the group consisting of talc, bentonite, silica, starch and maltodextrin.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions, in which the peptide of General formula (I) is represented in the drug selected from the group consisting of creams, emulsions of oil and/or silicone in water emulsions, water in oil and/or silicone oils, milk, balsams, foams, lotions, gels, liniments, sera, Soaps, ointments, foams, mousses, bars, pencils and sprays.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions, in which the peptide of General formula (I) are included in the solid substrate selected from the group consisting of wet wipes, hydrogels, adhesive patches, non-adhesive patches and masks.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions, in which the peptide of General formula (I) are included in the Cagnes, preferably in the form of bandages.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions containing the peptide of General formula (I), included in cosmetic products selected from the group consisting of masking means, tonal creams, lotions, makeup remover and face lotion, eye shadows and lipsticks.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions containing the peptide of General formula (I) in a concentration of between 0,00000001 wt.% and 20 wt.%.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions, in which the peptide of General formula (I) preferably is present in a concentration of between 0.0001 wt.% and 5 wt.%.

According to another important aspect of the present invention relates to cosmetic or pharmaceutical compositions containing additional cosmetically or pharmaceutically effective amount of the active component selected from the group consisting of exfoliating agent, moisturizing agent, depigmenting agent or a bleaching agent, a Pro-pigmenting agent, agent anti-wrinkle agent that can reduce or eliminate bags under the eyes, antioxidant, ant is pikirujushego agent, inhibitor of NO-synthase, agent, anti-aging agent stimulating the synthesis of dermal or epidermal molecules and/or for preventing their degradation, agents stimulating the proliferation of fibroblasts and/or keratinocytes or stimulating the differentiation of keratinocytes, agent for relaxing the skin, sealing agent, the agent against atmospheric pollution and/or against free radicals, agents acting on capillary circulation and/or microcirculation, soothing agent, an anti-inflammatory agent, agent, affect the metabolism of cells, organic or mineral photoprotective agent that is active against ultraviolet a and/or b rays, and mixtures thereof. The active agent preferably is either synthetic or plant extracts, or is the product of biofermentation.

According to another important aspect, the present invention relates to cosmetic or pharmaceutical compositions, in which the agent anti-wrinkle and/or anti-aging selected from the group consisting of Argireline®, Leuphasyl®, Decorinyl®, Decorinol®, Lipochroman®and Aldenine®sold Lipotec.

According to an important aspect, the present invention relates to the use of the peptide of formula (I) or its cosmetically or pharmaceutically acceptable salts of the otoplenie cosmetic or pharmaceutical compositions regulating neuronal exocytosis.

According to another important aspect, the present invention relates to the use of the peptide of formula (I) or its cosmetically or pharmaceutically acceptable salts in the manufacture of cosmetic or pharmaceutical compositions for the treatment, purification or skin care.

According to another important aspect, the present invention relates to the use of the peptide of General formula (I) or its cosmetically or pharmaceutically acceptable salts in the manufacture of cosmetic or pharmaceutical compositions for reducing and/or eliminating wrinkles on the face and/or facial asymmetry

According to another important aspect, the present invention relates to the use of the peptide of General formula (I) or its cosmetically or pharmaceutically acceptable salts in the manufacture of cosmetic or pharmaceutical compositions for reducing and/or eliminating facial wrinkles on the face.

According to another important aspect, the present invention relates to the use of the peptide of General formula (I) or its cosmetically or pharmaceutically acceptable salts in the manufacture of cosmetic or pharmaceutical compositions for reducing and/or eliminating facial wrinkles on the face by a local application on the forehead, the space between the eyebrows and/or wrinkles and face lines around the mouth and/or around the hole.

According to another important aspect, the present invention relates to the use of the peptide of General formula (I) or its cosmetically or pharmaceutically acceptable salts in the manufacture of cosmetic or pharmaceutical compositions for reducing and/or eliminating facial wrinkles on the face through the use of iontophoresis on the forehead, the space between the eyebrows and/or wrinkles and face lines around the mouth and/or eye.

According to another important aspect, the present invention relates to the use of the peptide of General formula (I) or its cosmetically or pharmaceutically acceptable salts in the manufacture of cosmetic or pharmaceutical compositions for reducing and/or eliminating facial wrinkles on the face through the use of subcutaneous or intradermal injection on the forehead, the space between the eyebrows and/or wrinkles and face lines around the mouth and/or eye.

According to another important aspect, the present invention relates to the use of the peptide of General formula (I) or its cosmetically or pharmaceutically acceptable salts in the manufacture of cosmetic or pharmaceutical compositions for reducing and/or eliminating muscle spasticity.

1. The peptide of General formula (I)
R1-AA-R2,
its stereoisomers, mixtures thereof and its cosmetically and pharmaceutically acceptable salts, characterized in, is that AA is a sequence 6-40 neighboring amino acids, contained in the amino acid sequence of SEQ ID No.1, selected from the group consisting of MAEDADMRNELEEMQRRADQL, ADESLESTRRMLQLVEESKDAGI, ELEEMQRRADQLA, ELEEMQRRADQL, ELEEMQRRADQ, ELEEMQRRAD, ELEEMQRRA, ELEEMQRR, LEEMQRRADQL, LEEMQRRADQ, LEEMQRRAD, LEEMQRRA, LEEMQRR, EEMQRRADQL, EEMQRRADQ, EEMQRRAD, EEMQRRA, EEMQRR, LESTRRMLQLVEE, NKDMKEAEKNLT, KNLTDL, IMEKADSNKTRIDEANQRATKMLGSG, SNKTRIDEANQRATKMLGSG, TRIDEANQRATKMLGSG, DEANQRATKMLGSG, NQRATKMLGSG and QRATKMLGSG;
R1selected from the group consisting of H or acetyl, or a saturated or unsaturated, linear, branched or cyclic With3-C24acyl, or polietilenglikoli polymer

where n can vary from 1 to 5, and
R2selected from the group consisting of amino, unsubstituted or substituted saturated linear or branched, or cyclic With1-C24aliphatic groups,
provided that when R1represents H or acetyl, then R2is not unsubstituted amino.

2. Cosmetic composition having inhibitory activity against neuronal exocytosis and containing a cosmetically effective amount of at least one peptide of General formula (I) according to claim 1, characterized in that it contains at least one cosmetically acceptable excipient or adjuvant.

3. Cosmetic composition according to claim 2, wherein the peptide of General formula (I) are included in the cosmetically acceptable system replaced the slow release or in the media, selected from the group consisting of liposomes, millicapsules, microcapsules, nanocapsules, sponges, vesicles, micelles, mallister, microspheres, nanospheres, lipofen, microemulsions, nanoemulsions, milicast, microparticles and nanoparticles.

4. Cosmetic composition according to claim 2, wherein the peptide of General formula (I) adsorbed on cosmetically acceptable substrate of the organic polymer or solid mineral substrate selected from the group consisting of talc, bentonite, silica, starch or maltodextrin.

5. Pharmaceutical composition having inhibitory activity against neuronal exocytosis and containing a pharmaceutically effective amount of at least one peptide of General formula (I) according to claim 1, characterized in that it contains at least one pharmaceutically acceptable excipient or adjuvant.

6. The pharmaceutical composition according to claim 5, wherein the peptide of General formula (I) are included in the pharmaceutically acceptable system slow release or in a carrier selected from the group consisting of liposomes, millicapsules, microcapsules, nanocapsules, sponges, vesicles, micelles, mallister, microspheres, nanospheres, lipofen, microemulsions, nanoemulsions, milicast, microparticles and nanoparticles.

7. The pharmaceutical composition according to claim 5, wherein the peptide of General formula is (I) adsorbed on a pharmaceutically acceptable substrate of the organic polymer or solid mineral substrate, selected from the group consisting of talc, bentonite, silica, starch or maltodextrin.

8. The use of the peptide of General formula (I) according to claim 1 in the manufacture of a cosmetic composition having inhibitory activity against neuronal exocytosis.

9. The use of the peptide of General formula (I) according to claim 1 in the manufacture of cosmetic compositions for the treatment, purification or skin care by reducing and/or eliminating facial wrinkles on the face.

10. The use of claim 8, which reduces and/or eliminates wrinkles on the face and/or facial asymmetry.

11. The use of the peptide of General formula (I) according to claim 1 in the manufacture of a pharmaceutical composition having inhibitory activity against neuronal exocytosis.

12. The application of item 11, which reduces and/or eliminates muscle spasticity.



 

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FIELD: chemistry.

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FIELD: medicine.

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19 cl, 7 tbl, 18 ex

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FIELD: medicine.

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FIELD: chemistry.

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FIELD: chemistry.

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9 cl, 12 tbl, 31 ex, 3 dwg

FIELD: chemistry.

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4 cl, 8 ex, 3 tbl, 1 dwg

FIELD: chemistry.

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23 cl, 75 dwg, 43 ex, 5 tbl

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13 cl, 7 dwg, 7 tbl, 5 ex

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6 cl, 3 dwg, 4 ex

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1 dwg, 4 ex

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