Peptides and their derivatives which interact with nicotinic acetylcholine receptor and suitable for use in cosmetology against mimic and age-related wrinkles

FIELD: biotechnology.

SUBSTANCE: compounds have the general formula: X1-X2-X3-Pro-X4-Pro-X5, where X1 is selected from H, Ac-, Palm-; X2 is selected from Trp, Tyr; X3 is selected from Trp, Tyr; X4 is selected from Lys, Orn, Dbu, Dpr, Arg; X5 is selected from-OH, -NH2, -OCH3, -OC2H5, -NH-C6H5. This compound can be represented in the form of retroisomer.

EFFECT: invention enables to block selectively the muscle nicotinic acetylcholine receptor with high efficiency.

12 cl, 7 dwg, 1 tbl, 5 ex

 

Peptides and derivatives thereof that interacts with the nicotinic acetylcholine receptor and is suitable for use in cosmetology against mimic and age-related wrinkles.

The technical field to which the invention relates.

The present invention relates to biochemistry, namely to new peptide compounds capable of selectively block muscle-type acetylcholine receptor. More specifically this invention relates to the use of such compounds in cosmetics for wrinkles and age-related wrinkles.

The level of technology.

In recent decades there has been a steady increase in life expectancy of the population of developed countries. In 2000, in the U.S. people over 65 years was 13%, in 2030 expect that this value will increase to 20%. This demographic shift requires intensified efforts to create effective medicines for people in older age groups. This requirement is fully applicable to cosmetic dermatology, which creates a firming, anti-aging and sun tools, and cream from wrinkles. Cosmetic and pharmaceutical companies often use such creams peptides as active ingredients. Peptides and cosmetics based on them have different activity: stimulation of fibroblast activity, inhibition of enzymes, p is prosaude collagen, stimulation of angiogenesis, immunomodulirutaya, regulation of melanin synthesis, blocking neuromuscular transmission. One of the most important obstacles for topical application of peptides in the creams is their reduced ability to penetrate the skin. In General, the ability of penetration depends on various factors: physical and chemical properties of matter (the dissociation constant of the acid [pKa], molecular size, stability, solubility and rate of lipophilicity); time of penetration; integrity, thickness and composition of the skin, skin metabolism; place, space, and duration of application (Ranade, V.V. Drug delivery systems. 6. Transdermal drug delivery. J. Clin. Pharmacol. 1991, 31, 401-418). Consider that the peptide is suitable for topical application if it meets the following options, but it's worth noting that this empirical parameters that are not universal (Guy, R.H. Current status and future prospects of transdermal drug delivery. Pharm. Res. 1996, 13, 1765-1769):

1. Molecular weight less than 500 Da

2. The value of the coefficient of lipophilicity (logarithm of the distribution coefficient in the system octanol/water) from 1 to 3

3. Melting point below 200 C

4. Good solubility in water (1 mg/ml)

5. No or little polar centers.

The peptides used in cosmetics, can be divided into four large groups: signal peptides, inhibit the market of enzymes Transporter peptides and peptide inhibitors of neuromuscular transmission (Gorouhi F, Maibach HI. Role of topical peptides in preventing or treating aged skin. Int J Cosmet Sci. 2009 Oct; 31(5): 327-45). Despite the same apparent physiological effect when using peptides from these four groups, the mechanism of action varies. Thus, the peptides of the group of blockers neuromuscular transmission are considered as a safe alternative to botulinum toxin injections in the fight against age and facial wrinkles. Having a high specificity, Botox, however, is not devoid of drawbacks, the main of which is the high toxicity and, as a consequence, the need for accurate calculation of the dose, a significant dependence of the quality of the drug from the conditions of production, use only in injectable form.

Muscle contraction is a physiological process by which muscles experience stress - are shortened or lengthened, thereby producing mechanical work. This process provides the ability of animals and humans to voluntary and involuntary movements and is directly related to food, respiratory, defensive, excretory and other physiological processes. Smooth muscles are responsible for involuntary movements, such as peristalsis of the stomach and intestines, changing the tone of blood soudo and bladder. Striated muscles provides random movement - a movement, facial expressions, breathing, swallowing, and other work of the heart is provided by the contraction of the heart muscles.

Muscles formed a multi-core muscle fibers, each of which separately is not only cellular, and physiological unit, which is due to the presence of such a specific "contractile" element as myofibrils. These fibers are combined in the beams of the first order; a few of these primary beams are connected, forming tufts of the second order, etc. before the formation of muscles. Because muscle contraction is caused by impulses from the Central nervous system, there are areas that are innervated by the synaptic endings of neuronal axons. The junction between a neuron and a muscle fiber is called the neuromuscular synapse (NMS).

The mechanism of muscle contraction can be divided into several main stages. The first is a transfer from neuron stimulus in the form of the action potential. When the action potential is propagated along the nerve fibers until his endings of muscle fibers that leads to the release of the neurotransmitter acetylcholine (ach) from the presynaptic part of the NMS in the synaptic gap. This neurotransmitter acts to limit the military region of the membrane of the muscle fiber (postsynaptic part NMS), opening multiple managed acetylcholine channels - acetylcholine receptor (AHR). The result of the discovery channel is the increase in the concentration of sodium ions inside the muscle fibers that leads to the membrane action potential, which is conducted along the membrane of the muscle fiber. The action potential depolarizes muscle membrane, which leads to the selection of sarcoplasmatic reticulum large number of calcium ions stored in it. Calcium ions directly initiate the process of muscle contraction. In the future, using the calcium pump in the membrane sarcoplasmatic reticulum calcium ions are pumped back, leading to relaxation of the muscles.

The mechanism of functioning of the neuromuscular synapse is described in sufficient detail. When the nerve impulse, expressed in the form of an action potential arrives at the end of the motor neuron, it causes to be revealed in the presynaptic membrane NMS calcium channels. Local increase here the intracellular concentration of calcium ions promotes their interaction with proteins, which contribute to the fusion of synaptic vesicles filled with neurotransmitter AH, with the plasma membrane of a neuron. The last process is also studied in detail and by means of the SNARE complex formed by the effective the effective chetyrehstvolnym the interaction of the three proteins synaptobrevin from the surface of the vesicles, syntaxin and SNAP-25 with the surface of the membrane of a neuron. The formation of this complex leads to rapid fusion of vesicular and plasma membranes and causes exocytosis AH in the synaptic gap. The acetylcholine molecules diffuse through the slit width of 50-100 nm and reaches the postsynaptic membrane, which has a high sensitivity to the mediator due to the presence of high-affinity receptors AHR. Linking AH causes the opening of the canal; this forms a strong gradient of sodium ions into the cell, but much weaker flow of potassium ions out with the subsequent depolarization of the muscle membrane, the release of calcium and muscle contraction, as stated above.

The mechanism of transmission of nerve impulse through the postsynaptic part of the NMS is mediated through the interaction of the neurotransmitter AH with AHR and also studied in detail. There are two large groups of AHR - nicotine (Nahr) and muscarinic (machr), which differ in their ability to bind specific agonists. So, first got its name because of the high affinity to the plant alkaloid nicotine, and the latter - to the alkaloid from poisonous mushrooms to muscarine. Nahr are ionotropic receptors, that is a classic mnogozaryadnymi ion channels that open DL the passage of certain ions in the binding of the ligand. mahr refers to metabotropic receptors; this is a single-chain protein with 7 transmembrane fragments, coupled with G-protein. In this case, the transmission signal after binding of the ligand is due to the large number of metabolic pathways.

At the molecular level Nahr are oligomeric proteins composed of 5 subunits. It is known that all 5 subunits are located in the membrane pseudosymmetric around the Central axis, which forms an ion channel with a diameter of approximately 2.5 nm. These data were obtained for Nahr from the electric organ of Torpedo rays and having a subunit stoichiometry (α1)2-β1-γ-δ [Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J Mol Biol 2005; 346: 967-89]. To date, all identified 10 different subtypes of α-subunits (α1-α10) and 4 subtype β-subunits (β1-β4). All of these subtypes of α - and β-subunits (except for α1 - and β1-) found in the neuronal subtypes Nahr located mainly in neurons of the Central and/or peripheral nervous systems, in many cases also on the presynaptic membrane [Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol 2007; 47: 699-729]. Postsynaptic Nahr neuromuscular synapse animals and humans have stoichiometry (α1)2-β1-γ-δ (similar to that for the receptor from the electric organ) only at the initial embryonic) stage of development of the organism. In the adult form of the γ-subunit is substituted on the ε- [Yomoto N., Wakatsuki, S., Sehara-Fujisawa, A. 2005. The acetylcholine receptor gamma-to-epsilon switch occurs in individual end plates. Biochem. Biophys. Res. Commun. 331: 1522-1527]. Nahr structure (α1)2-β1-ε-δ and is the only form of Nahr in adult muscle, responsible for the reception of neurotransmitter, causing as a result, the reduction of this muscle. Given the high homology of all subunits Nahr give them the same pentamers channel spatial organization in the membrane. Today it is known also about the location of the binding sites of classical agonists and competitive antagonists at Nahr: two ligand-binding site are located in the contact regions of large N-terminal extracellular domains of two α1 and neighbouring γ(ε) and δ subunits of the receptor around their middle part with respect to the membrane surface.

To achieve fast transfer of information between neuron and muscle fiber requires high concentrations of AHR in the right areas postsynaptic membrane NMS. Therefore, clustering AHR is one of the most important stages in the process the proper functioning of NMS [Hoch W. 1999. Formation of the neuromuscular junction. Agrin and its unusual receptors. Eur. J. Biochem. 265: 1-10]. It involved several proteins, the most important of which are agrin, Rapson and kinase MuSK (Muscle-Specific Kinase). Designed to present the time and created today new agents, blocking neuromuscular transmission, aimed at disrupting the normal functioning or presynaptic membrane NMS or postsynaptic side of the synapse.

The cosmetic industry has made several attempts to develop new compounds for topical application in the treatment of facial wrinkles, in order to avoid adverse effects observed at the injection botanicheskogo toxin (Lupo MP, Cole AL. Cosmeceutical peptides. Dermatol Ther. 2007, Sep-Oct; 20(5): 343-9). To date, there are several peptide compounds that block neuromuscular transmission.

Table 1.
Amino acid sequences of peptides that block neuromuscular transmission, and their biological target.

NameAmino acid sequenceTarget
Argireline extractAc-Glu-Glu-Met-Gln-Arg-Arg-NH2Inhibits SNARE complex formation and release of neurotransmitter.
LamasilH-Tyr-D-Ala-Gly-Phe-Leu-OH Mimics the action enkefalina - reduces the excitation of the neuron
VialoxH-Gly-Pro-Arg-Pro-Ala-NH2Competitive antagonist mahr
SinacH-β-Ala-Pro-Dab-NHBzlCompetitive antagonist mahr
InulinHe publishedCompetitive antagonist MuSK

First in the list of "cosmetic analogues of Botox" is the peptide Argireline extract (Blanes-Mira, S., et al. A synthetic hexapeptide (Argireline) with antiwrinkle activity. Int J Cosmet Sci. 2002; 24(5): 303-310). Appearing in the early 2000's, he quickly gained popularity today can be found in many cosmetic products, aims to combat facial wrinkles. Six amino acids Argireline repeat SNAP25 protein site required for binding of the synaptic vesicle axon to the presynaptic membrane. In AXONE Argireline extract competes with protein SNAP25 and built instead a temporary SNARE protein complex - this complex is formed of several membrane proteins immediately before the binding of the vesicle with the membrane and is required for successful exocytosis. Defective complex is unable to provide the necessary contact of the bubble with memb the Ana, the result is not a release of neurotransmitter in the synaptic gap and the muscle does not receive the signal on the reduction and continues to be in a relaxed state.

Analogues Argireline obtained from the protein SNAP-25 and directed to the inhibition of neuromuscular transmission at the synaptic level, due to competition with SNAP-25 for the formation of the SNARE complex, described in patent EP 1180524 and WO 9734620.

For the volunteers it was shown that topical application of a cream containing 10% Argireline, within 30 days, resulting in 30% decrease in the depth of facial wrinkles versus 10% for placebo.

Pentapeptide Lavutil - development company Lipotec (Spain), mimics the action enkefalina - reduces the excitation of the neuron, inhibiting the flow of calcium ions through the membrane, and reduces Ca2+-dependent release of neurotransmitter. Application Lafazia mixed with Argireline leads to a strengthening of the effect of the latter in 1.5.

Inulin - acetyl Hexapeptide developed by company Lipotec (Spain) (WO 2011/009626), created with the help of molecular modeling, in accordance with the predicted structure of the binding site agrin in MuSK (Muscle-Specific Kinase). Inulin acts as a competitive antagonist of MuSK in the binding site agrin, which is essential for muscle contraction. Postsynaptic mechanism to reduce muscle contractions allows huts in order to avoid the formation of facial wrinkles. Tests on human volunteers showed that the use of a 5% solution inline within 28 days reduces the depth of wrinkles in the crow's feet at 14.9%.

Vialox (Vialox, the company Pentapharm, Switzerland) - Pentapeptide, which is the fragment component of the venom of the temple Viper-ballerina-1. Vialox has a curare-like activity and is a competitive antagonist of acetylcholine receptor. When the peptide is bound to the receptor sodium ion channel is in the closed position, which interrupts the passage of nerve impulses and prevents muscle decline.

Patent EP 1809652 describes the peptide antagonists of the acetylcholine receptor, which act on postsynaptic mechanism, similar to the action valerina-1 - block nerve transmission and prevent the formation of wrinkles. Vialox can be used in combination with other cosmetic peptides (WO 2006/069608).

When using a cream with 5% content of vialox within 28 days leads to size reduction of wrinkles by 49%, and roughness of the skin by 47%.

One cosmetic active peptide, Sinak (Syn-ake, the company Pentapharm, Switzerland) is a reversible antagonist of the muscular nicotinic acetylcholine receptor (mahr). This Tripeptide acts by a mechanism similar to the action of valerina-1, binds to the Epsilon subunit of the th mahr and thus, preventing the binding of acetylcholine and channel activation. Therefore, the muscles remain in a relaxed state (WO 2006/047900). Tests on human volunteers showed that the application of a cream with 4% Syn-Ake within 28 days reduces the depth of wrinkles in the forehead by 52%.

Thus, the most effective peptides to reduce facial wrinkles are Vialox and Sinac - competitive antagonists mahr.

However, no connection, developed cosmetic or pharmaceutical industry is not able to inhibit muscle contraction with efficiency similar to that of botulinum toxin. Therefore, there is still a need for new compounds able to inhibit muscle contraction to achieve the best results in reducing and softening of wrinkles and, in particular, facial wrinkles.

Disclosure of the invention.

Definition.

In the present description refer to amino acids correspond to the rules recommended by the IUPAC-IUB (Eur. J. Biochem., 1984, 138: 9-37; J. Biol. Chem., 1989, 264: 633-673). For example, Lys mean NH2-CH(CH2-CH2-CH2-NH2)-COOH, Lys - mean NH2-CH(CH2-CH2-CH2-NH2)-CO-, -Lys means-NH-CH(CH2-CH2-CH2-NH2)-COOH, -Lys - means-NH-CH(CH2-CH2-CH2-NH2)-CO-. Therefore, feature, on nachusa a peptide bond, removes the hydroxyl carboxyl group of the amino acids, when located to the right of the symbol amino acids, and the proton alpha-amino group of amino acids, when located to the left of the amino acid symbol.

The term Ac denotes acetyl group (CH3-CO-) and Palm - means Palmitoyl group (CH3-(CH2)14-CO-).

The term Orn represents the amino acid ornithine, Dbu-2,4-diaminobutane acid, Dpr-2,3-diaminopropionic acid, -NH-C6H5- benzylated.

The term "cosmetically acceptable salts" of the peptides in the context of the present invention, means salts acceptable for use in humans and include salts formed by addition of inorganic bases, such as lithium, sodium, calcium, potassium, magnesium, copper, zinc, etc. or organic bases, for example, ethylamine, diethylamine, Ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, or a salt obtained by adding organic acids such as acetate, citrate, lactate, malonate, maleate, fumarate, benzoate, aspartate, glutamate, oleate, triptorelin, oxalate, gluconate, or inorganic acids, for example, chloride, sulfate, borate, carbonate. Cosmetically acceptable salts of the peptides of the present invention can be obtained by standard methods (S.M. Berge, Bighley L.D. and Monkhouse, D.C. 1977, "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

The present invention is ESET the task of expanding Arsenal of peptides-blockers neuromuscular transmission suitable for use in cosmetology and dermatology.

The closest analogues on the mechanism of action of the claimed peptides are peptides Sinac and Vialox. Both peptides developed by the Swiss company "Pentapharm" based component of the venom of the temple Viper valerina-1, which contains 21 amino acid. It is known that valerin-1 blocks of muscle Nahr, that is their antagonist (Weinstein, S.A., et al. Characterization and amino acid sequences of two lethal peptides isolated from the venom of Wagler''s pit viper, Trimeresurus wagleri. Toxicon. 1991; 29(2): 227-36). Short peptides based on it does not have toxicity, and retain the ability in high concentrations block the same receptor. Recently (Utkin Y.N. et al. Azemiopsin from Azemiops feae venom Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor. J. Biol. Chem. 2012, Aug 3; 287(32): 27079-86) in the venom of the Burmese Viper Azemiops feae venom was opened polypeptide-azemiopsin containing 21 amino acids (DNWWPKPPHQGPRPPRPRPKP) and is also able to block muscle Nahr. Thus azemiopsin more active compared to valerina-1.

During the search of the active centre of azemiopsin (plot amino acid sequence that defines a major contribution to the ability to communicate with Nahr) using methods electrophysiology (Figure 1) was tested panel intersecting pentapeptidnogo portions of the source of the toxin (Figure 2). Examined their ability to inhibit induced by acetylcholine currents through expressed in oocytes, Spa zeway Xenopus laevis muscle Nahr. Preliminary testing of these fragments was carried out at concentration of 150 µg/ml. the Results of this study are shown in figure 2. Most active in this panel was Pentapeptide SEQ ID NO:1:WWPKP. However, this peptide does not show toxicity intraperitoneal injection at doses up to 30 mg/kg, and when administered orally in doses up to 160 mg/kg.

To study the influence of the structure of the peptide SEQ ID NO:1:WWPKP on its biological activity in the sequence of the peptide (hereinafter Az3) were amended: 1. entered amide group at the C-end (SEQ ID NO:41: (H-Trp-Trp-Pro-Lys-Pro-NH2, further Az3-NH2); 2. substituted residues tryptophan to tyrosine residues: [Tyr1]Az3 (SEQ ID NO:3), [Tyr2]Az3 (SEQ ID NO:2) and [Tyr1,Tyr2]Az3 (SEQ ID NO:4); and replacement of the second amino acid residue for a hydrophobic residue valine ([Val-2]Az3); 4. replacement of lysine-4 diaminobutane acid SEQ ID NO:5: (H-Trp-Trp-Pro-Dbu-Pro-OH (next [Dbu4]Az3); 5. the sequence of the peptide was reduced to four and three amino acid residues: [des-Trp1]Az3, [des-Pro5]Az3, [des-Trp1,des-Pro5]Az3; 6. the obtained variants of the peptide SEQ ID NO:1 by substitution of lysine-4 on arginine: WYPRP-NH2([Tyr2,Arg4]Az3) and YYPRP-NH2([Tyr1,Tyr2 Arg4]Az3); 7. the resulting peptides with various modifications of the carboxyl group Pro5: methyl ether and benzylated: WYPKP-OCH3Ac-YYPKP-och3Ac-WYPKP-NH-C6H5, YYPKP-NH-C6H5. P the results of the research of biological activity of the obtained variants of the peptide SEQ ID NO:1 is shown in Figure 3 and 7. Studied the ability to suppress induced by acetylcholine currents through expressed in Xenopus oocytes Xenopus laevis muscle Nahr. Testing of these fragments was carried out at concentrations of 1 mm. We can conclude that most of the obtained modified peptides retained biological activity in relation to muscle Nahr, except [Val-2]Az3, [des-Trp1]Az3, [des-Pro5]Az3, [des-Trp1,des-Pro5]Az3.

Synthesized and tested retroperitoneally peptides Az3 and [Tyr2]Az3: PKPWW and PKPYW respectively. Inhibitory activity in relation to muscle receptor saved, but is at a lower level. Figure 5 shows the diagram of the test retroposition and comparison with the activity of the source Az3 and [Tyr2]Az3. The peptides were tested at a concentration of 1 mm.

To compare the efficiency of suppression of the current through the channel of the receptor fragments azemiopsin and commercial samples of Sinaica and Vialox were conducted additional experiments. The substance was tested at a concentration of 1 mm. The results are shown in Figure 3.

According to the results of the tests most appropriate was considered in future studies to use amidinophenoxy form pentapeptidnogo movie azemiopsin with the replacement of the second amino acid to tyrosine SEQ ID NO:42: (H-Trp-Tyr-Pro-Lys-Pro-NH2(next [Tyr2]Az3-NH2).

For a more detailed comparison [Tyr2]Az3-NH2 with the more common commercial product Synacom curves were constructed based inhibitory activity on concentration which are presented in figure 3. The concentration at which there is a suppression of the current at 50% (IC50 value)for peptide [Tyr2]Az3-NH2 corresponds to 23 μm, whereas a similar concentration of Senaka is equal to 180 μm, that is, a new peptide was almost 8 times functionally more active than a commercial product. This is reflected in the histogram in figure 5.

Compounds of the invention.

1. The task of obtaining new compounds blocking mahr is solved at the expense of the structure of a peptide having the following formula (I): X1-X2-X3-Pro-X4-Pro-X5, where

X1 is chosen from H, Ac-, Palm-

X2 is selected from Trp, Tyr,

X3 is selected from Trp, Tyr,

X4 is chosen from Lys, Orn, Dbu, Dpr, Arg

X5 is selected from-OH, -NH2, -OCH3, -OC2H5, -NH-C6H5,

Preferably the compounds of formula (I) are selected from the group consisting of:

SEQ ID NO:1: (H-Trp-Trp-Pro-Lys-Pro-OH

SEQ ID NO:2: N-Trp-Tyr-Pro-Lys-Pro-OH

SEQ ID NO:3: H-Tyr-Trp-Pro-Lys-Pro-OH

SEQ ID NO:4: (H-Tyr-Tyr-Pro-Lys-Pro-OH

SEQ ID NO:5: (H-Trp-Trp-Pro-Dbu-Pro-OH

SEQ ID NO:6: (H-Trp-Tyr-Pro-Dbu-Pro-OH

SEQ ID NO:7: H-Tyr-Trp-Pro-Dbu-Pro-OH

SEQ ID NO:8: H-Tyr-Tyr-Pro-Dbu-Pro-OH

SEQ ID NO:9: Ac-Trp-Trp-Pro-Lys-Pro-OH

SEQ ID NO:10: Ac-Trp-Tyr-Pro-Lys-Pro-OH

SEQ ID NO:11: Ac-Tyr-Trp-Pro-Lys-Pro-OH

SEQ ID NO:12: Ac-Tyr-Tyr-Pro-Lys-Pro-OH

SEQ ID NO:13: Ac-Trp-Trp-Pro-Dbu-Pro-OH

SEQ ID NO:14: Ac-Trp-Tyr-Pro-Dbu-Pro-OH

SEQ ID NO:15: Ac-Tyr-Trp-Pro-Dbu-Pro-OH

SEQ ID NO:16: Ac-Tyr-Tyr-Pro-Dbu-Pro-OH

SEQ ID NO:17: Ac-Trp-Trp-Pro-Lys-Pro-NH2

SEQ ID NO:18: Ac-Trp-Tyr-Pro-Lys-Pro-NH2

SEQ ID NO:19: Ac-Tyr-Trp-Pro-Lys-Pro-NH2

EQ ID NO:20: Ac-Tyr-Tyr-Pro-Lys-Pro-NH 2

SEQ ID NO:21: Ac-Trp-Trp-Pro-Dbu-Pro-NH2

SEQ ID NO:22: Ac-Trp-Tyr-Pro-Dbu-Pro-NH2

SEQ ID NO:23: Ac-Tyr-Trp-Pro-Dbu-Pro-NH2

SEQ ID NO:24: Ac-Tyr-Tyr-Pro-Dbu-Pro-NH2

SEQ ID NO:25: (H-Trp-Trp-Pro-Lys-Pro-OCH3

SEQ ID NO:26: (H-Trp-Tyr-Pro-Lys-Pro-OCH3

SEQ ID NO:27: (H-Tyr-Trp-Pro-Lys-Pro-OCH3

SEQ ID NO:28: (H-Tyr-Tyr-Pro-Lys-Pro-OCH3

SEQ ID NO:29: (H-Trp-Trp-Pro-Dbu-Pro-OCH3

SEQ ID NO:30: (H-Trp-Tyr-Pro-Dbu-Pro-OCH3

SEQ ID NO:31: (H-Tyr-Trp-Pro-Dbu-Pro-OCH3

SEQ ID NO:32: H-Tyr-Tyr-Pro-Dbu-Pro-OCH3

SEQ ID NO:33: Ac-Trp-Trp-Pro-Lys-Pro-OCH3

SEQ ID NO:34: Ac-Trp-Tyr-Pro-Lys-Pro-OCH3

SEQ ID NO:35: Ac-Tyr-Trp-Pro-Lys-Pro-OCH3

SEQ ID NO:36: Ac-Tyr-Tyr-Pro-Lys-Pro-OCH3

SEQ ID NO:37: Ac-Trp-Trp-Pro-Dbu-Pro-OCH3

SEQ ID NO:38: Ac-Trp-Tyr-Pro-Dbu-Pro-OCH3

SEQ ID NO:39: Ac-Tyr-Trp-Pro-Dbu-Pro-OCH3

SEQ ID NO:40: Ac-Tyr-Tyr-Pro-Dbu-Pro-OCH3

SEQ ID NO:41: (H-Trp-Trp-Pro-Lys-Pro-NH2

SEQ ID NO:42: (H-Trp-Tyr-Pro-Lys-Pro-NH2

SEQ ID NO:43: (H-Tyr-Trp-Pro-Lys-Pro-NH2

SEQ ID NO:44: (H-Tyr-Tyr-Pro-Lys-Pro-NH2

SEQ ID NO:45: (H-Trp-Trp-Pro-Dbu-Pro-NH2

SEQ ID NO:46: (H-Trp-Tyr-Pro-Dbu-Pro-NH2

SEQ ID NO:47: (H-Tyr-Trp-Pro-Dbu-Pro-NH2

SEQ ID NO:48: (H-Tyr-Tyr-Pro-Dbu-Pro-NH2

The peptides of the present invention may consist of stereoisomers or mixtures of stereoisomers of amino acids, i.e. amino acids can be L - and/or D-configuration, or to be racemic mixture, independently of each other.

The technical result of the claimed invention is achieved due to such properties of the new peptide, such as: selective blocking of MNH is, the lack of toxicity and allergenicity.

Because the claimed peptides are small and contain 5 amino acid residues, they can be obtained by chemical synthesis. The inventive peptides are novel compounds and have no close homologues.

The inventive peptides can be used in cosmetics in the cosmetic composition in the form of a cream, lotion or gel for application to the skin to reduce the depth of expression and age-related wrinkles. The weight concentration of the peptide in the cosmetic composition is in the range of 0.01-5%. In addition to the compounds of General formula (I) such cosmetic composition may contain at least one additional component selected from: amino acids, proteins, hydrolyzed proteins, growth factors, enzymes, enzyme inhibitors, peptides, oligosaccharides, polysaccharides, pyrimidines, purines and nucleotides, nucleosides, carboxylic acids, fatty acids, lipids, sphingolipids, flavonoids, phenols, polifenoles, terpenes, alkaloids, benzofuranol, polyalcohol, an antimicrobial component, antimicrobial peptides, vitamins, provitamins, retinoids, carotenoids, chelating agents, antioxidants, substances which improve the permeability of the skin.

The invention is illustrated Figure 1-6:

Figure 1 is an example of a write current through the muscle the AHR, heterological expressed in Xenopus oocytes Xenopus laevis, in response to the addition of 20 μm acetylcholine. Each application of acetylcholine, in the presence of ligand or without marked with a black rectangle on top. Control the current to the far left is compared to the current obtained in the presence of 50 μm of azemiopsin. The arrow shows the start time is inactivated. The time interval between applications of acetylcholine 5 minutes. Demonstrated complete suppression of the azemiopsin activity of the muscle receptor.

Figure 2. Comparison of the effectiveness of suppression of current through the channel muscle Nahr overlapping five-membered fragments of azemiopsin, as well as commercially available compounds Syn-ake and Vialox. 100% made complete suppression of the current 0% to the current level of control. All connections are taken at a concentration of 150 µg/ml.

Figure 3. Comparison of the effectiveness of suppression of current through the channel muscle Nahr various derivatives of azemiopsin and commercially available drugs Syn-ake and Vialox. The tested compounds were taken in a concentration of 1 mm. 100% made complete suppression of the current 0% to the current level of the control current in response to application of 20 μm acetylcholine.

Figure 4. Comparison of curves inhibitory activity concentration for peptide SEQ ID NO:41 ([2Tyr]Az3NH2) and commercial product Syn-ake. The abscissa axis sediments is s decimal logarithms of the concentrations of the ligands, the ordinate axis deferred current through the channel of the receptor in percent of the control current in response to application of 20 μm acetylcholine.

Figure 5. Comparison of the concentrations of half-suppression of the response of the current through the channel muscle Nahr. The ordinate axis deferred concentration of the test drug ([Tyr2]Az3-NH2 and Syn-ake).

6. Comparison of the effectiveness of suppression of current through the channel muscle Nahr peptides Az3-NH2, [2Tyr]Az3-NH2 and their retroperitoneally concentration of 1 mm. 100% made complete suppression of the current 0% to the current level of control.

7. Write current through muscle Nahr, heterological expressed in Xenopus oocytes Xenopus laevis, in response to the addition of 20 μm acetylcholine. Each application of acetylcholine, in the presence of ligand or without marked with a black square on top. Control the current to the far left is compared to the current obtained in the presence of 1 mm solution of the peptide. The grey rectangles indicate the period of application of the test peptide. For peptide WYPKP-OCH3(Figa) inhibition of the current was 40%for Ac-YYPKP-OCH3(Figb) inhibition of the current was 38%for Ac-WYPKP-NH-C6H5(Pigv) inhibition of the current was 62%, for YYPKP-NH-C6H5(Figg) inhibition of the current was 18%, for WYPRP-NH2(Figd) inhibition of the current was 100%, for YYPRP-NH2(Fige) inhibition current is made 100%;

Examples of embodiment of the invention.

Example 1. Chemical synthesis of the peptide SEQ ID NO:1: (H-Trp-Trp-Pro-Lys-Pro-OH.

Attaching the first amino acid. Obtaining Fmoc-Pro-P (Fmoc=9-fluorenylmethoxycarbonyl, P=polymer).

200 mg of 2-chloroethylamino polymer with a content of hydroxyl groups of 1.0 mmol/g) was washed with anhydrous methylene chloride. In 5 ml of methylene chloride is dissolved 374 mg (1.1 mmol) Fmoc-Pro-OH and 374 μl (2.2 mmol) of diisopropylethylamine, the solution is stirred for 5 min and then poured to the polymer. The reaction is performed for 1 hour at room temperature and stirring. Upon completion of the reaction the polymer is filtered off and washed three times successively methylene chloride, ethyl alcohol, dimethylformamide.

Description one synthetic cycle capacity polypeptide chain.

Obtaining Fmoc-Lys(Boc)-Pro-P:

(a) Obtained in the previous step peptidyl-polymer for 20 min, treated with 20%solution of piperidine in dimethylformamide. The polymer is washed successively with 5 ml of the following solvents: dimethylformamide - 3 times for 2 min, the mixture of dioxane-water (2:1) 2 times for 5 min, dimethylformamide - 5 times in 2 minutes

b) In 5 ml of dimethylformamide is dissolved 505 mg (1.1 mmol) of Fmoc-Lys(Boc)-OH, 150 mg (1.1 mmol) of 1-hydroxybenzotriazole, and 170 μl (1.1 mmol) of N,N'-diisopropylcarbodiimide, the solution is stirred 10 min at 0°C and then poured to poly the ERU. The reaction is performed for 4 hours with periodic mixing. Upon completion of the reaction, the polymer is filtered off, washed with dimethylformamide, and treated with 5 ml of a mixture of Ac2O-pyridine-dimethylformamide (20:20:60) for 1 h, after which the polymer was successively washed with dimethylformamide, isopropanol, dimethylformamide.

Synthesis of polypeptide chains carried out manually in a glass flow reactor (2×20 cm) according to the following Protocol for each synthetic cycle (at the rate of 8-10 ml of solvent to 400 mg of the original polymer), when carrying out the condensation reaction (step 6) use the volume of the reaction mixture 5-7 ml:

1. DMF (dimethylformamide) (5×2 min);

2. 20% piperidine in DMF (20 min);

3. DMF (3×2 min);

4. dioxane-water, 2:1, (2×5 min);

5. DMF (5×2 min);

6. The reaction of condensation: 5 molar equivalents of activated Fmoc-amino acids (4 o'clock);

7. DMF (3×2 min);

8. acylation: Ac2O-pyridine-dimethylformamide, 20:20:60, (1 o'clock);

9. DMF (3×2 min);

10. isopropanol (3×2 min);

For activation of Fmoc-derivatives of amino acids using a mixture of DIPCDI/HOBT (N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole) to a solution of 1.1 mmol (5 equivalents) of Fmoc-protected amino acids and 150 mg (1.1 mmol, 5 equivalents), HOBt in 4 ml of DMF add 170 μl (1.1 mmol, 5 equivalents) of DIPCDI, the solution is stirred for 10 minutes

Completeness of the reaction is ondensate control using ninhydrin or in the case of the N-terminal Proline, Satinover tests after surgery 6 synthetic Protocol.

For synthesis use the following derivatives of amino acids: Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH.

Cleavage of the peptide from the polymer.

For the reaction of cleavage of the peptide from the resin and simultaneous release of the protective groups of the side chains of the amino acids taken 800 mg of the peptidyl-resin. To peptidyl-polymer is poured 15 ml of a mixture of TFU (triperoxonane acid) -H2O in volumetric ratio of 97.5:2.5, the suspension is stirred for 2 hours, then the resulting peptide solution was filtered from the polymer washed with 5 ml of TFU and excess TFU evaporated under reduced pressure. The peptide is precipitated with 100 ml of ethyl ether, filtered and washed with ether (5×20 ml). The residue is dissolved in 5 ml of 10% acetic acid for 20 min, filtered and washed with 5 ml of 10% acetic acid. The resulting solution of the peptide lyophilizer and absoluut in column (a 2.5×60 cm) with Sephadex G-10 in 0.1M acetic acid. Purification of the peptide is carried out using reversed-phase HPLC in a gradient of acetonitrile (10% to 35% for 75 min) in 0.1% acetic acid at a flow rate of eluent 3 ml/min, the eluate absorbance recorded at a wavelength of 226 nm. Fractions corresponding to the main peak in the chromatogram collected and lyophilizers. The molecular mass of the peptide as set by the method of mass-spectrum of the industry, was 713.8 Yes, calculated theoretical molecular weight of 712.8 Yes.

Example 2. Determination of acute toxicity of peptide H-Trp-Trp-Pro-Lys-Pro-OH.

Determination of acute toxicity (LD50) intraperitoneal injection of peptide H-Trp-Trp-Pro-Lys-Pro-OH was carried out on 40 male mice of Balb weighing 20-23 g, divided into five equal groups. The peptide was administered to mice intraperitoneally at a concentration of 1 mg/kg, 3 mg/kg, 10 mg/kg and 30 mg/kg in 0.2 ml of sterile 0.9% NaCl solution. Animals of the control group in the same volume was injected with 0.9% NaCl solution. According to the results of the experiment found no toxic effect of the peptide in any of these concentrations.

Study of acute toxicity (DI50per os) with the introduction of the peptide in the stomach conducted on 40 male mice of Balb weighing 20-23, Animals were randomly divided into 5 equal groups. The drug was administered to the animals once in the stomach by using a special probe in doses of 2.5 mg/kg, 10 mg/kg, 40 mg/kg, 160 mg/kg in 0.2 ml of sterile 0.9% NaCl solution. Animals of the control group in the same volume was injected with 0.9% NaCl solution. With the introduction of the investigated peptide in the stomach of mice in the above concentrations was not observed toxic effect.

Example 3. Preparation of a cosmetic cream containing peptide H-Trp-Trp-Pro-Lys-Pro-OH in the range of the weight concentrations of 0.01-5%. The composition of the REM: H-Trp-Trp-Pro-Lys-Pro-IT - 0.01%, 1% or 5%, glycerol 20%, and stearic acid - 2%, butylparaben - 1%, glyceryl stearate 1%, Cetearyl alcohol - 2%, decillia - 3%, water to 100%. All ingredients, except water and peptide H-Trp-Trp-Pro-Lys-Pro-OH, are mixed and heated to 70°C to obtain a homogeneous transparent mass. The mixture is then cooled to a temperature of 40°C and added dropwise with vigorous stirring an aqueous solution of the peptide H-Trp-Trp-Pro-Lys-Pro-OH. Allow the cream to cool slowly to room temperature.

Example 4. The study of skin-irritating action of the drug in acute, single impact, and subacute, repeated daily exposure for 2 weeks, the experiments were carried out on five healthy volunteers (3 women (age 24, 29 and 51) and 2 men (age 44, 46 years). Samples of the cream containing 0.01%, 1% or 5% of the investigated peptide was applied daily for two weeks on the forehead and forearm. Skin irritation was not observed either in a single or sequential application of the drug.

Example 5. Electrophysiological study of the conductivity of nicotinic acetylcholine receptors (Nahr) muscle type, heterological expressionary in Xenopus oocytes Xenopus laevis. Adult clawed frog (not less than 8 cm) were placed in a solution of para-aminobenzoic acid for 15-20 minutes until fully immobilized. To retrieve not the required number of oocytes did the incision in the skin and a layer of muscles on the belly of a frog parallel to the "bikini line" and separated a small portion of the ovary. The extracted part of the ovary was placed in ND96 buffer without calcium ions, and crushed. If necessary, the advanced oocytes were treated with collagenase solution. Operated animal sewn operating cuts tissue with suture material from polyglycolide. Each clawed frog operated not more often than once in 6 weeks.

Receptor expression was performed in accordance with the following procedure: a solution of a mixture of plasmids containing inserts protestirovannyx genes α1, β1, δ and ε-subunits of muscle Nahr under control of the CMV-promoter, were injected with in the core of freshly isolated oocytes by setting Nanoject ("Drummond scientific, USA), after which the oocytes were incubated at 18°C for 48 hours.

Nahr muscle type is the ligand-operated ion channel. When interacting with the agonist (acetylcholine or nicotine) receptor undergoes a conformational change, which through time between its subunits are positively charged ions. When connecting the electrodes to the membrane of cells expressing this receptor, it is possible to write the ionic current through open channels receptors.

Oocytes were placed in a cell with electrodes connected to the amplifier Turbo-TEC H (NPI Electronics, USA); the camera was pre-filled with ND96 buffer. Testing was conducted on Utah by two-electrode of fixed potential on the membrane. The electrodes of the amplifier is made from borosilicate glass and filled with 3M KCl was injected into the oocyte. Membrane potential was recorded at - 70 mV. Oocytes expressing muscle Nahr, was subjected to 20 μm acetylcholine every five minutes up until the amplitude of the current in response to application of acetylcholine did not reach a stationary level. Then there was the five minute preincubation oocyte in the solution of the test peptide. After a five-minute inactivated with peptide oocyte was subjected to solution 20 μm acetylcholine in the presence of the test ligand. The experiments were repeated with different concentrations of test peptides in multiple oocytes from different parties. The amplitude of the current in response to application of acetylcholine in the presence of the test ligand was compared with the amplitude of the current in response to the control application (in the absence of test ligand)that preceded each experience. To clarify the characteristics of the test peptide built the dependence of the current amplitude (expressed as percentage of control) of the decimal logarithm of the concentration of the ligand. The calculation of the inhibition curves and values AS was performed using the program Origin7.5 using the model "dose response".

1. The compound of General formula (I): X1-X2-X3-Pro-X4-Pro-X5, where
X1 is chosen from H, Ac-, Palm-
X2 is selected from Trp, Tyr,
X3 is selected from Trp, Ty,
X4 is chosen from Lys, Orn, Dbu, Dpr, Arg
X5 is selected from-OH, -NH2, -OCH3, -OC2H5, -NH-C6H5,
characterized by the ability to selectively block the muscle nicotinic acetylcholine receptor and is suitable for use in cosmetic for wrinkles and age-related wrinkles, and this connection can be represented as retrogamer.

2. The compound according to claim 1, wherein X1 means Ac-

3. The compound according to claim 1, characterized in that the X5 means-NH2

4. The compound according to claim 1, wherein X2 and X3 means-Trp or Tyr-, X4 means-Lys - or-Dbu-.

5. The compound according to claim 1, characterized in that it has the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48

6. The application of one of the compounds according to any one of claims 1 to 5 as an active substance for blocking neuromuscular transmission.

7. The application of one of the compounds according to any one of claims 1 to 5 to obtain cosmetic compositions for topical application, p is egodnoe for wrinkles and age-related wrinkles.

8. Cosmetic composition for wrinkles and age-related wrinkles, characterized in that it contains as active principle a compound of General formula (I) in the weight range of concentrations from 0.01 to 5%.

9. The cosmetic composition of claim 8, characterized in that it contains: at least one compound of General formula (I) and at least one additional component selected from amino acids, proteins, hydrolyzed proteins, growth factors, enzymes, enzyme inhibitors, peptides, oligosaccharides, polysaccharides, pyrimidines, purines and nucleotides, nucleosides, carboxylic acids, fatty acids, alcohols, lipids, sphingolipids, flavonoids, terpenes, alkaloids, benzofuranol, polyalcohol, an antimicrobial component, antimicrobial peptides, vitamins, provitamins, retinoids, carotenoids, chelating agents, antioxidants, substances improving the permeability of the skin.

10. The cosmetic composition of claim 8 for wrinkles and age-related wrinkles, characterized in that it contains dermatologically acceptable carrier.

11. The cosmetic composition of claim 8, wherein, in the form of solution, dispersion, emulsion or liposome.

12. The cosmetic composition of claim 8, characterized in that it presents in the form of a lotion, jelly, ointment, cream, gel, gel-like polymers, SP the EEV or facial masks.



 

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