KR20230039143A - Peptides with improved transdermal drug delivery efficacy and lipolysis - Google Patents

Abstract

It relates to a peptide with improved transdermal drug delivery efficacy and lipolytic ability, and the modified peptide according to one aspect improves the transdermal delivery efficacy of the peptide through a change in net charge and has a lipolytic effect, so it can be useful for preventing or treating obesity. there is.

Description

Peptides with improved transdermal drug delivery efficacy and lipolysis

It relates to a peptide with improved transdermal drug delivery efficacy and lipolytic ability.

Obesity is a very serious disease that is rapidly increasing in many countries around the world but has no medically effective treatment. The World Health Organization (WHO) defines a body mass index (BMI) of 30 or more as obesity, and the average obesity rate in OECD member countries is 19.5%, and the rate is gradually increasing.

Obesity refers to the state of excessive accumulation of fat in the body due to genetic or lifestyle causes, and causes diseases such as adult diseases and chronic degenerative diseases. are doing Secondary complications such as hyperlipidemia, hypertension, arteriosclerosis, diabetes, and fatty liver caused by excessive fat accumulation and social disorders that can be caused by obesity are problems.

Recently, various peptides having a function of enhancing or inhibiting the activity of enzymes have been developed because they are structurally simpler than proteins as polymers of amino acids, but have a very large interaction with proteins in vivo.

Despite high biocompatibility and physiological activity, peptides have low bioavailability, limiting their use. In particular, effective delivery of peptides was difficult due to the barrier function of the skin, which is famous for the 500 Dalton's Law. The average molecular weight of one amino acid is about 100 daltons, and peptides composed of 5-100 amino acids cannot effectively pass through the skin barrier.

Existing methods for transdermal delivery of peptides include binding cell penetrating peptides (CPPs) or fatty acids to the peptides.

However, in the method of binding cell-permeable peptides or fatty acids to existing peptides with high physiological activity, there are problems such as reduction in physiological activity due to structural changes in peptides that may occur in the reaction process and increase in production cost accompanying additional coupling reactions. Disadvantages do exist.

Accordingly, the present inventors continued research on transdermal delivery using peptides and, as a result, synthesized a new peptide with excellent skin penetration and permeability and excellent lipolysis effect.

In one aspect, beta-melanocyte stimulating hormone (beta-Melanocyte stimulating hormone; β-MSH) peptide containing the amino acid sequence represented by SEQ ID NO: 1 is modified, the modified peptide at the C-terminus or N-terminus of the amino acid sequence is to provide

Another aspect is to provide a cosmetic composition for lipolysis comprising the modified peptide as an active ingredient.

Another aspect is to provide a pharmaceutical composition for preventing or treating obesity comprising the modified peptide as an active ingredient.

Another aspect is to provide a health functional food for preventing or improving obesity containing the modified peptide as an active ingredient.

Another aspect includes modifying the C-terminal or N-terminal amino acid sequence of beta-melanocyte stimulating hormone (β-MSH) comprising the amino acid sequence represented by SEQ ID NO: 1 , To provide a method for producing a modified peptide.

In one aspect, beta-melanocyte stimulating hormone (beta-Melanocyte stimulating hormone; β-MSH) peptide containing the amino acid sequence represented by SEQ ID NO: 1 is modified, the modified peptide at the C-terminus or N-terminus of the amino acid sequence to provide.

The beta-melanocyte stimulating hormone (beta-Melanocyte stimulating hormone; β-MSH) is an endogenous peptide hormone and neuropeptide, and is a peptide naturally present in the human body. It is also known to have an effect of reducing appetite.

The beta-melanocyte stimulating hormone may have 5 to 100 amino acid residues including the amino acid sequence represented by SEQ ID NO: 1.

The term "amino acid" herein may include the 20 standard amino acids that are naturally incorporated into peptides as well as D-isomers and modified amino acids.

The peptide may be D-type or L-type, a peptide in which only a portion of the sequence is D-type or L-type, or a racemate thereof.

The peptide may be conservatively substituted at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in a native or unmutated peptide, but is not limited thereto.

As used herein, the term “conservative substitution” refers to the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. The peptide may have, for example, one or more conservative substitutions while still retaining the biological activity of the native or unmutated peptide. Such amino acid substitutions can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; Negatively charged (acidic) amino acids include glutamic acid and aspartic acid; Aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In addition, amino acids can be classified into amino acids with electrically charged side chains and amino acids with uncharged side chains, and amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, Amino acids with uncharged side chains, including arginine and histidine, can be further classified as nonpolar amino acids or polar amino acids, and nonpolar amino acids are glycine, alanine, valine, leucine, and isoleucine. Methionine, proline, and polar amino acids can be classified as including serine, threonine, cysteine, asparagine, and glutamine. Conservative substitutions with amino acids having similar properties as described above can be expected to exhibit the same or similar activity.

In addition, the peptide may include post-translational modification of non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including eg acetylation, myristoylation and palmitoylation), amidation ( amidation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g. beta -removal deimidation, deamidation) and structural changes (eg formation of disulfide bridges). In addition, it may include changes in amino acids caused by chemical reactions occurring in the process of bonding with crosslinkers to form a peptide conjugate, such as changes in amino groups, carboxy groups, or side chains. .

In one embodiment, the modification of the amino acid sequence is aspartic acid (D), glutamic acid (Glutamic acid, E), lysine (K), arginine (Arginine, R) and histidine (Histidine, H). Any one or more selected from the group consisting of beta-may be added to or deleted from the end of the melanocyte stimulating hormone. Here, the term "terminal" does not mean one amino acid at the most end of the C-terminus or N-terminus of the peptide, but may mean up to 5 amino acids from the most end of the C-terminus or N-terminus. . In addition, the amino acid sequence may be modified by adding it together with other amino acids without being limited to the types of amino acids described above. In this case, the addition or deletion of the amino acid may not affect the original functionality of beta-melanocyte stimulating hormone.

Even if it is described as a 'peptide consisting of a specific sequence number' in this specification, if it has the same or corresponding activity as the peptide consisting of the amino acid sequence of the sequence number, the addition of meaningless sequences before and after the amino acid sequence of the sequence number or natural Mutations that may occur due to, or silent mutations thereof are not excluded, and it is obvious that even in the case of having such sequence additions or mutations, they fall within the scope of the present invention. That is, even if there is a difference in some sequences, as long as they show a certain level of homology and exhibit beta-melanocyte stimulating hormone activity, they may fall within the scope of the present invention. Specifically, the peptide is 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84% of the amino acid sequence of SEQ ID NO: 1 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99% homology Or it may include an amino acid sequence having the same identity, but is not limited thereto.

“Homology” or “identity” means the degree to which two given amino acid sequences or base sequences are related to each other and can be expressed as a percentage. The terms “homology” and “identity” are often used interchangeably.

Whether any two peptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: can be determined using known computer algorithms such as the “FASTA” program using default parameters as in 2444. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453). (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215] : 403 (1990);Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073) Homology, similarity or identity can be determined using, for example, BLAST of the National Center for Biotechnology Information Database, or ClustalW.

Homology, similarity or identity of peptides can be found in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482; for example, Needleman et al. (1970), J Mol Biol. 48: 443. It can be determined by comparing sequence information using the GAP computer program. In summary, the GAP program defines the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (i.e., amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or 10 gap opening penalty, 0.5 gap extension penalty); and (3) no penalty for end gaps. Thus, as used herein, the terms "homology" or "identity" indicate relevance between sequences.

In one embodiment, the modified peptide can be prepared by a combination of several methods for preparing various peptides.

The modified peptide of the present invention can be synthesized by a method well known in the art, for example, an automated peptide synthesizer, or can be produced by genetic engineering technology. Specifically, the modified peptides of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other art method. Thus, modified peptides according to the present invention can be synthesized in a number of ways, including, but not limited to, for example:

(a) synthesizing peptides stepwise or by fragment assembly by means of solid-phase or liquid-phase methods, and isolating and purifying the final peptide product; or

(b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or

(c) a method of performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

A method of obtaining a peptide fragment by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

In addition, preparation of the modified peptides may include modification using L- or D-type amino acids and/or non-natural amino acids; and/or modification of the native sequence, e.g., modification of side chain functional groups, intramolecular covalent bonds, e.g., inter-side chain ring formation, amidation, methylation, acylation, ubiquitination, phosphorylation, aminohexaylation, biotinylation, etc. It includes all that transform by reforming. In addition, the above modifications include all substitutions with non-natural compounds.

Substituted or added amino acids used in the modification may be 20 amino acids commonly observed in human proteins as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids may include, but are not limited to, Sigma-Aldrich, ChemPep and Genzyme pharmaceuticals. Peptides containing these amino acids and typical peptide sequences may be synthesized and purchased through commercialized peptide synthesis companies, such as American Peptide Company or Bachem in the US, or Anygen in Korea, but are not limited thereto.

According to one embodiment, the present inventors deleted the N-terminal glutamic acid and C-terminal aspartic acid of beta-melanocyte stimulating hormone and further deleted N-terminal alanine to prepare a modified beta-melanocyte stimulating hormone.

In one embodiment, as described above, from the group consisting of Aspartic acid (D), Glutamic acid (E), Lysine (K), Arginine (R) and Histidine (H) By adding or deleting one or more selected ones to the terminal of the beta-melanocyte stimulating hormone, the absolute value of the net charge of the beta-melanocyte stimulating hormone may be increased to 1.5 or more. Transdermal delivery efficacy of the peptide can be improved through such a change in the net charge amount. Specifically, by modifying the amino acid sequence as described above to increase the absolute value of the net charge to 1.5 or more, the efficacy of transdermal delivery of peptides by diffusion or microcurrent, for example, iontophoresis, may be improved. . The peptide having an increased net charge as described above may have a higher transdermal permeability due to microcurrent than through passive diffusion. Accordingly, the modified peptide by modification of the amino acid sequence may have improved transdermal delivery efficacy compared to conventional beta-melanocyte stimulating hormone.

In one embodiment, the modified peptide may be a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.

In one embodiment, the modified beta-melanocyte stimulating hormone peptide may have a lipolytic effect, and as the transdermal delivery efficacy is increased through the modification as described above, it is more effectively transdermally delivered than before the modification, so that the lipolytic effect is increased. may have been elevated.

In one embodiment, the present inventors confirmed that the modified beta-melanocyte stimulating hormone peptide as described above has improved transdermal delivery efficacy and lipolytic effect.

As used herein, the term "iontophoresis" refers to a method of allowing ionized active ingredients to penetrate the skin with electrical repulsive force by changing the electrical environment of the skin by applying a potential difference by flowing a microcurrent through the skin to which the active ingredient is applied means The iontophoresis is a method in which a current from an external power source flows into an electrode patch on the skin and a microcurrent is introduced into the skin, a method in which a battery is attached to the electrode patch itself and a microcurrent is introduced into the skin, and a high concentration It may include a method in which microcurrent is introduced into the skin through a patch equipped with a reversed electrodialysis (RED) means for generating current through a difference in ion concentration between the electrolyte solution and the low-concentration electrolyte solution. However, the present invention is not limited thereto, and various types of iontophoresis can be used, of course. In addition, the device causing iontophoresis is at least one battery selected from the group consisting of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry battery, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electrodialysis battery. or may be a second mask pack, mask sheet, or patch in which the at least one battery is mounted. In one embodiment, by modifying the amino acid sequence of the beta-melanocyte stimulating hormone peptide to increase the absolute value of the net charge to 1.5 or more, it was confirmed that the transdermal permeability of the peptide was improved by generating a microcurrent by iontophoresis. .

In one embodiment, the beta-melanocyte stimulating hormone peptide may not be a cell-penetrating peptide (CPP).

In the present specification, the term "cell penetrating peptide (CPP)" is a cell membrane penetrating peptide, and is generally classified into cationic and amphipathic α-helix according to physicochemical properties. Cationic CPP has a high net charge, especially a high proportion of arginine (R). Amphiphilic α-Helix CPP is known to exist in the form of a random coil in a hydrophilic environment such as the cytoplasm, but to have an α-Helix form with hydrophilic and hydrophobic surfaces when exposed to a hydrophobic environment such as a cell membrane.

The beta-melanin cell stimulating hormone peptide may not be a cell penetrating peptide and have a high molecular weight, so that the percutaneous permeability before modification may be low.

Another aspect provides a cosmetic composition for lipolysis comprising the modified peptide as an active ingredient.

The cosmetic composition includes a cosmetically effective amount of the peptide; And / or may include a cosmetically acceptable carrier.

As used herein, the term "cosmetic effective amount" means an amount sufficient to achieve the lipolysis effect of the cosmetic composition.

The cosmetic composition may be prepared in any formulation commonly prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil , It may be formulated as a powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. For example, it may be formulated into a softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components It can be.

When the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, for example, in the case of a spray, additionally chlorofluoro propellants such as hydrocarbons, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspension agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the cosmetic composition is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid Amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Ingredients included in the cosmetic composition include ingredients commonly used in cosmetic compositions, in addition to peptides and carrier components as active ingredients, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. A phosphorus adjuvant may be included.

Another aspect provides a pharmaceutical composition for preventing or treating obesity comprising the modified peptide as an active ingredient.

As used herein, the term "pharmaceutical composition" can refer to a molecule or compound that imparts some beneficial effect upon administration to a subject. Beneficial effects may include enabling diagnostic determination; amelioration of a disease, symptom, disorder or condition; reducing or preventing the occurrence of a disease, condition, disorder or condition; and responding to a disease, symptom, disorder or condition in general. In the present invention, the pharmaceutical composition may give a beneficial effect on obesity or metabolic diseases.

As used herein, the term “prevention” refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its attendant symptoms, preventing the acquisition or re-acquisition of a disease or disorder, or preventing the occurrence or recurrence of a disease or disorder. means of reducing the risk of acquisition. For example, the prevention refers to any action that inhibits or delays the occurrence of obesity or metabolic disease symptoms by administration of the composition according to the present invention.

As used herein, the term "treatment" includes inhibition, alleviation or elimination of the development of a disease, such as obesity or a metabolic disease.

As used herein, the term "improvement" may refer to any activity that at least reduces a parameter associated with alleviation or treatment of a condition, for example, the degree of obesity or metabolic disease symptoms.

The "metabolic disease" may refer to a disease caused by excessive synthesis or accumulation of fat due to abnormal energy metabolism in the body due to various causes such as excessive energy intake or hormonal imbalance. Thus, metabolic diseases can be caused by obesity. The type of the metabolic disease is not particularly limited, but may be at least one selected from the group consisting of fatty liver, diabetes, hypertension, hyperlipidemia, hypercholesterolemia, dyslipidemia, and arteriosclerosis.

In one embodiment, the modified peptide may be in the form of a pharmaceutically acceptable salt thereof. The salts are conventional acid addition salts used in the field of medicine, for example obesity or metabolic diseases, for example salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid and acetic acid, propionic acid , succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, oxalic acid or trifluoroacetic acid It includes salts derived from organic acids such as In addition, the salt may be a base addition salt such as ammonium, dimethylamine, monomethylamine, monoethylamine, or diethylamine. In addition, the salt includes a common metal salt form, for example, a salt derived from a metal such as lithium, sodium, potassium, magnesium, or calcium. The acid addition salt, base addition salt or metal salt may be prepared according to a conventional method. Pharmaceutically acceptable salts and general methodologies for preparing them are well known in the art. See, for example, P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)]; [S.M. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, no. 1, January 1977].

For the condensation of the protected amino acid or peptide, various activating reagents useful in peptide synthesis, particularly preferably triphosphonium salt, tetramethyluronium salt, carbodiimide and the like can be used. Examples of triphosphonium salts include benzotriazol-1-yloxytris(pyrrolazino)phosphonium hexafluorophosphate (PyBOP), bromotris(pyrrolazino)phosphonium hexafluorophosphate (PyBroP), 7 -Including azabenzotriazol-1-yloxytris(pyrrolazino)phosphonium hexafluorophosphate (PyAOP), examples of tetramethyluronium salts include 2-(1H-benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro Phosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(5-norbonane-2,3 -Dicarboxyimide)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), O-(N-succimidyl)-1,1,3,3-tetramethyluronium tetrafluoro Roborate (TSTU), examples of carbodiimides include N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIPCDI), N-ethyl-N '-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl); For condensation using these, racemization inhibitors [e.g., N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB), 1-hydroxybenzotriazole (HOBt), 1-hydroxy- 7-azabenzotriazole (HOAt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), ethyl 2-cyano-2-(hydroxyl addition of mino)acetate (Oxyma, etc.) is preferred. The solvent used for the condensation can be appropriately selected from those known to be useful for peptide condensation reactions. For example, acid amides such as anhydrous or water-containing N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc., halogenated hydrocarbons such as methylene chloride, chloroform, etc.; Alcohols such as fluoroethanol and phenol, sulfoxides such as dimethyl sulfoxide, tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, methyl acetate, ethyl Esters such as acetate and the like, suitable mixtures thereof, and the like can be used. The reaction temperature is appropriately selected from a range known to be usable for the peptide bonding reaction, and is usually selected from the range of about -20°C to 90°C. Activated amino acid derivatives are usually used in excess of 1.5 to 6 fold. In the solid phase synthesis, when a test using a ninhydrin reaction indicates that the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If condensation is still insufficient after repeating the reaction, the unreacted amino acid can be acetylated with an acid anhydride, acetylimidazole or the like, so that the influence on the subsequent reaction can be avoided.

Examples of protecting groups for the amino group of the starting amino acid are benzyloxycarbonyl (Z), tert-butoxycarbonyl (Boc), tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl , 2-chlorobenzyloxycarbonyl (Cl-Z), 2-bromobenzyloxycarbonyl (Br-Z), adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitro phenylsulfenyl, diphenylphosphinothioyl, 9-fluorenylmethyloxycarbonyl (Fmoc), trityl and the like.

Examples of carboxyl-protecting groups for starting amino acids include aryl, 2-adamantyl, 4-nitrobenzyl, 4-methoxy, in addition to the C1-6 alkyl groups, C3-10 cycloalkyl groups, and C7-14 aralkyl groups mentioned above. benzyl, 4-chlorobenzyl, phenacyl and benzyloxycarbonylhydrazide, tert-butoxycarbonylhydrazide, tritylhydrazide and the like.

The hydroxyl groups of serine or threonine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include lower (C2-4) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, and groups derived from organic acids and the like. Additionally, examples of groups suitable for etherification include benzyl, tetrahydropyranyl, tert-butyl (But), trityl (Trt), and the like.

Examples of protecting groups for the phenolic hydroxyl group of tyrosine include Bzl, 2,6-dichlorobenzyl, 2-nitrobenzyl, Br-Z, tert-butyl, and the like.

Examples of protecting groups for imidazole of histidine are p-toluenesulfonyl (Tos), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), dinitrophenyl (DNP), benzyloxymethyl (Bom), tert-butoxymethyl (Bum), Boc, Trt, Fmoc, and the like.

Examples of protecting groups for the guanidino group of arginine are Tos, Z, 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), p-methoxybenzenesulfonyl (MBS), 2,2, 5,7,8-pentamethylchroman-6-sulfonyl (Pmc), mesitylene-2-sulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Includes phonyl (Pbf), Boc, Z, NO2 and the like.

Examples of protecting groups for side chain amino groups of lysine include Z, Cl-Z, trifluoroacetyl, Boc, Fmoc, Trt, Mtr, 4,4-dimethyl-2,6-dioxocyclohexylidenyl (Dde), and the like. includes

Examples of protecting groups for indolyl of tryptophan include For, Z, Boc, Mts, Mtr, and the like.

Examples of protecting groups for asparagine and glutamine include Trt, xantyl (Xan), 4,4'-dimethoxybenzhydryl (Mbh), 2,4,6-trimethoxybenzyl (Tmob), and the like.

Examples of activated carboxyl groups in the starting materials include corresponding acid anhydrides, azides, active esters [esters with alcohols such as pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol , cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccimide, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt))], etc. include Examples of activated amino groups in the starting material include the corresponding phosphorus amide.

Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon; Anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid (TFA), trimethylsilyl bromide (TMSBr), trimethylsilyl trifluoromethanesulfonate, tetrafluoroboric acid, tris(tri acid treatment with a solution of fluoro)boric acid, boron tribromide, or a mixture thereof; Base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc.; and reduction with sodium in liquid ammonia; and the like. The elimination reaction by acid treatment described above is generally carried out at a temperature of -20°C to 40°C; Acid treatments include anisole, phenol, thioanisole, metacresol and paracresol; This is done efficiently by adding a cation scavenger such as dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol, triisopropylsilane, and the like. In addition, the 2,4-dinitrophenyl group used as the protecting group of the imidazole of histidine is removed by thiophenol treatment; The formyl group used as a protecting group for the indole of tryptophan is obtained not only by acid treatment in the presence of 1,2-ethanedithiol and 1,4-butanedithiol, but also by alkali treatment with dilute sodium hydroxide and dilute ammonia. removed by deprotection.

Protection of a functional group not to be involved in the reaction between the starting material and the protecting group, removal of the protecting group, activation of a functional group involved in the reaction, and the like can be appropriately selected from known protecting groups and known means.

For the peptides mentioned herein, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to conventional peptide markings. The C-terminus of the peptide may be any one of an amide (-CONH2), a carboxyl group (-COOH), a carboxylate (-COO-), an alkylamide (-CONHRa), and an ester (-COORa).

In the method for preparing an amide of a peptide, it is formed by solid phase synthesis using a resin for amide synthesis, or the α-carboxyl group of a carboxy terminal amino acid is amidated, and the peptide chain is extended toward the amino group side to a desired chain length. Then, a peptide from which the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a peptide with only the protecting group for the C-terminal carboxyl group removed from the peptide chain are prepared, and these two peptides are mixed as described above. condensed in a solvent. Regarding the details of the condensation reaction, the same as above applies. After the protected peptide obtained by condensation is purified, all protecting groups can be removed by the method described above to obtain the desired crude peptide. By purifying this crude peptide using various publicly known means of purification of the major fraction and freeze-drying, the desired amide of the peptide can be prepared.

In one embodiment, the modified peptide may be in the form of a solvate thereof. “Solvate” means that the peptide or salt thereof forms a complex with solvent molecules.

The pharmaceutical composition is a formulation for oral administration, a formulation for intraoral application, a formulation for administration by injection, a formulation for dialysis and irrigation, a formulation for application to the bronchi and lungs, a formulation for administration to the eyes, a formulation for administration to the ear, and a formulation for application to the nose. It may be any one formulation selected from the group consisting of formulations for application to the body, formulations for rectal application, formulations for vaginal application, and formulations for application to the skin.

The composition comprises a pharmaceutically effective amount of the modified peptide; and/or a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" may mean an amount sufficient to achieve the efficacy of preventing or treating obesity of the pharmaceutical composition.

The pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

When formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, liurine fat, glycerogeratin and the like may be used.

The pharmaceutical composition may include carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, and small molecules in order to increase stability or absorption. Proteins or other Stabilizers can be used as pharmaceuticals.

Another aspect provides a method for treating obesity or metabolic disease comprising administering the pharmaceutical composition to a subject.

As used herein, the term "administration" means providing a pharmaceutical composition to a subject by any suitable method.

A pharmaceutically effective amount and an effective dosage of the pharmaceutical composition may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition. In addition, the type and degree of response to be achieved by administration of the pharmaceutical composition, the type of subject to be administered, age, weight, general health condition, symptom or degree of disease, gender, diet, excretion, simultaneous or It may vary according to various factors including drugs and other components of the composition used together in this case, and similar factors well known in the medical field. A person of ordinary skill in the art can readily determine and prescribe an effective dosage for the desired treatment. Administration of the pharmaceutical composition according to the present invention can be administered once a day, divided into several administrations. Therefore, the dosage is not intended to limit the scope of the present invention in any way. The dosage of the pharmaceutical composition may be 1 ug/kg/day to 1,000 mg/kg/day.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual who needs a cure for obesity or metabolic disease or has a desire to lose weight.

Another aspect provides a health functional food for preventing or improving obesity containing the modified peptide as an active ingredient.

In this specification, the term "health functional food" refers to a food manufactured and processed by using a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, etc. a specific ingredient contained in a food raw material for the purpose of health supplement. It refers to food designed and processed so that it can sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. The health functional food composition may perform functions related to prevention and improvement of obesity and prevention and improvement of metabolic diseases.

There is no particular limitation on the type of food. Examples of foods to which the above substances may be added include powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, leached teas, and formulations selected from the group consisting of health drinks, etc. It includes all health foods in the usual sense.

The health beverage composition of the present invention may include various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

The health functional food may include food additives acceptable in food science, and may include appropriate carriers commonly used in the manufacture of health functional food.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages; and the like. In addition, the composition of the present invention may include fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01 to 0.1 part by weight per 100 parts by weight of the composition of the present invention.

Another aspect includes modifying the C-terminal or N-terminal amino acid sequence of a beta-Melanocyte stimulating hormone (β-MSH) peptide comprising the amino acid sequence represented by SEQ ID NO: 1 To provide a method for producing a modified peptide.

In one embodiment, the step of modifying the amino acid sequence is any one or more selected from the group consisting of Aspartic acid, Glutamic acid, Lysine, Arginine, and Histidine. It can be addition or deletion. In addition, the N-terminal alanine may be further deleted.

Among the terms or elements mentioned in the description of the modified peptide, the same as those already mentioned are as described above.

The modified peptide according to one aspect improves transdermal delivery efficiency of the peptide through a change in net charge and has a lipolytic effect, so it can be usefully used for preventing or treating obesity.

1 is a graph showing the results of CD (Cicular Dichroism) spectrum analysis of modified β-MSH peptide 2.
Figure 2 is a graph showing that the skin penetration depth of the peptide can be known from the fluorescence intensity.
Figure 3 is a CLSM (Confocal Laser Scanning Microscopy) analysis picture showing that the skin permeability of modified β-MSH peptides is improved compared to conventional β-MSH peptides.
Figure 4 is a graph showing the weight loss when the modified β-MSH peptide 2 (KKD peptide) was applied to obese mice.
Figure 5 is a photograph showing the weight loss when the modified β-MSH peptide 2 (KKD peptide) was applied to obese mice.
6 is a photograph showing that the size of inguinal white fat was reduced when modified β-MSH peptide 2 (KKD peptide) was applied to obese mice.
7 is a graph showing that the size of inguinal white fat decreased when modified β-MSH peptide 2 (KKD peptide) was applied to obese mice.
8 is a photograph showing that the size of adipose tissue is reduced when the modified β-MSH peptide 2 (KKD peptide) is applied to obese mice.

Hereinafter, the present invention will be described in more detail with examples, but this is only illustrative and is not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below may be modified within a range that does not deviate from the essential gist of the invention.

Example 1. Selection of peptide suitable for microcurrent

There are peptides under specific conditions in which skin permeability increases by microcurrent, and they are screened and shown in Table 1 below.

Conditions for Transdermal Delivery Molecular Weight structure net charge (z) Microcurrent strengthening effect One <800 CPP 1.5 <│z│ doesn't exist 2 1.5 >│z│ doesn't exist 3 No CPP 1.5 <│z│ doesn't exist 4 1.5 >│z│ doesn't exist 5 >800 CPP 1.5 <│z│ doesn't exist 6 1.5 >│z│ doesn't exist 7 No CPP 1.5 <│z│ has exist 8 1.5 >│z│ doesn't exist

As shown in Table 1, CPP (cell penetrating peptide, cell penetrating peptide) alone was effective in transdermal delivery, and the increase in transdermal delivery efficiency by RED (reverse electrodialysis, microcurrent generator) was insignificant. Even when the molecular weight was less than 800 Daltons, transdermal delivery was well achieved alone, and the increase in transdermal delivery efficiency by RED was insignificant. In addition, when the net charge amount was less than 1.5, the effect of the microcurrent was small, and the increase in transdermal delivery efficiency was insignificant.

The net charge of a peptide is the sum of the charges of each amino acid constituting the peptide, and has a great effect on its behavior in an electromagnetic field. Peptide molecules with a large absolute value of net charge receive strong force in the electromagnetic field, and peptide molecules with zero net charge are not affected by the electromagnetic field. In particular, since the net charge of a peptide changes according to pH, the behavior in the electromagnetic field varies according to the pH condition of the peptide solution.

Therefore, according to the above results, non-CPP peptides having a molecular weight greater than 800 Daltons and a large absolute value of net charge could be expected to increase transdermal delivery efficiency when using microcurrent.

Example 2. Peptide sequence design suitable for microcurrent

As shown in Example 1, when the sequence of a peptide that does not satisfy the specific conditions is modified to satisfy the conditions suitable for microcurrent, the skin permeability can be improved.

Amino acids that affect the net charge of the peptide are shown in Table 2 below. When aspartic acid (D) or glutamic acid (E) is added to the terminal of the parent peptide, the charge becomes -1, and lysine (K), arginine (R) or histidine (Histidine, When H) is added to the end of the parent peptide, the charge becomes +1. Accordingly, it can be seen that the transdermal transduction rate can be improved when microcurrent is used by modifying the parent peptide in such a way as to add or exclude the above amino acids from the terminus that does not affect the functionality of the parent peptide.

An example of this is shown in Table 3 below, and the peptide modified by adding a lysine amino acid to the end of the parent peptide, which is not suitable for the existing microcurrent, has a net charge of 2.0 or more, making it a suitable peptide for microcurrent. there is.

As shown in Table 4 below, using the design principle, β-MSH, a non-CPP peptide having a high molecular weight and a small net charge, was modified to increase the net charge. Specifically, by deleting glutamic acid (E) and alanine (A) at the N-terminus of the existing parent peptide β-MSH through the Solid Phase Peptide Synthesis (SPPS) method, the net charge is 0.1 A modified β-MSH peptide 1 increased to 1.1 was prepared. In addition, by deleting glutamic acid (E) and alanine (A) at the N-terminus of the existing parent peptide β-MSH and aspartic acid (D) at the C-terminus, the net charge was 0.1 A modified β-MSH peptide 2 increased to 2.1 was prepared.

Amino acid Net charge at pH 7.0 Basic amino acids Asp(D), Glu(E) -One Acidic amino acid Lys (K), Arg (R), His (H) One

Existing parent peptide Microcurrent Incompatible Peptide CPP structure MW (g/mol) Net charge (at pH 7.0) X 2000.0 0.0 Peptide modified by adding two Lys to the end of the parent peptide Microcurrent suitable peptide CPP structure MW (g/mol) Net charge (at pH 7.0) X 2256.3 2.0

Existing β-MSH peptide Modified β-MSH Peptide 1 Modified β-MSH Peptide 2 order MW (g/mol) Net charge (at pH 7.0) order MW (g/mol) Net charge (at pH 7.0) order MW (g/mol) Net charge (at pH 7.0) AEKDEGPYRMEHFRWGSPPKD (SEQ ID NO: 1) 2660.92 0.1 KKDEGPYRMEHFRWGSPPKD (SEQ ID NO: 2) 2460.73 1.1 KKDEGPYRMEHFRWGSPPK (SEQ ID NO: 3) 2345.64 2.1

Experimental Example 1. Secondary structure analysis of modified peptides

The secondary structure of β-MSH peptide 2 modified in Example 2 was analyzed.

The peptide structure was analyzed using a CD spectrometer to confirm the secondary structure and CPP characteristics of the peptide. This is because CD analysis can confirm the specific amphipathic alpha helix structure taken by CPP peptides or secondary structural changes due to amino acid sequence changes. CPP peptides are mainly classified as amphipathic CPP with an amphipathic alpha helical structure and random coil structure with a high ratio of arginine among constituent amino acids or a large net charge value (Cationic CPP). are classified

For the CD analysis condition, a solution in which trifluoroethanol (TFE), a peptide bending-inducing solvent, was mixed in a 1:1 volume ratio with 10 mM, pH 7.4, phosphate buffered saline, which had little interference with the UV measurement wavelength, was used. This is because the secondary structure formed by bending of the peptide is usually induced when a hydrophobic environment such as a cell membrane is provided instead of an aqueous solution.

As a result of confirming the structure of the existing β-MSH peptide and the modified β-MSH peptide, as shown in Table 5 and FIG. 1, modified β-MSH peptide 2 was confirmed to have a random coil-oriented secondary structure. Therefore, considering the structure of the existing β-MSH peptide and the modified β-MSH peptide, the low arginine ratio, and the low net charge amount, it can be seen that the CPP peptide has weak characteristics and is not a CPP peptide. Accordingly, the modified β-MSH peptide was not a CPP peptide, and the net charge amount was increased, so that the transdermal permeability could be increased as follows.

1 is a graph showing the results of CD (Cicular Dichroism) spectrum analysis of modified β-MSH peptide 2.

Secondary structural components Modified β-MSH Peptide 2 α-Helix (%) 24.2 β-sheet (%) 15.5 Turn (%) 18.4 Random(%) 41.9

Experimental Example 2. Confirmation of improvement in transdermal delivery efficiency of modified peptides

In order to confirm the improvement in the transdermal delivery efficiency of the modified β-MSH peptide in Example 2, a fluorescent substance (FITC) was chemically bound to the modified peptide, and then the results of the skin permeation test were analyzed with CLSM equipment.

For the skin permeation test, Franz Diffusion Cell, a representative in vitro skin absorption test method, was used. Hairless mouse (CrlOri:SKH1-hr, 8 week) was placed on the receiving compartment with the stratum corneum of the skin facing upward, and then a fluorescent substance was bound to the donor compartment. A peptide solution (50 μM, phosphate buffered saline) was added and microcurrent was applied (RED) or not applied (Passive Diffusion). The receiving compartment was filled with phosphate buffered saline, and the temperature was maintained at 32 ± 1 °C for 1 hour.

Percutaneous penetration depth analysis was performed using CLSM equipment, and tomography was taken at 5 μm intervals for a total of 150 μm from 20 μm on the upper skin to 130 μm on the lower skin in z-stack mode with the equipment set to the FITC wavelength band. As shown in FIG. 2, after quantifying the tomographic image using the Image-J program, the depth of skin penetration (d ₀ ) with the highest intensity of fluorescence is the same as the intensity of natural fluorescence on the skin surface. After setting to the maximum penetration depth (d _max ), it was calculated by the following formula.

d = d _max - d ₀

(d: skin penetration depth, d _max : maximum penetration depth, d ₀ : skin surface depth)

As a result of the skin permeation experiment, as shown in FIG. 3, the modified β-MSH peptide compared to the existing β-MSH peptide showed improved skin permeability when using microcurrent . This is because β-MSH is not a CPP and has a large molecular weight and a high net charge.

Figure 2 is a graph showing that the skin penetration depth of the peptide can be known by the fluorescence intensity.

Figure 3 is a CLSM (Confocal Laser Scanning Microscopy) analysis picture showing that the skin permeability of modified β-MSH peptides is improved compared to conventional β-MSH peptides.

Experimental Example 3. Confirmation of citrate synthase inhibitory ability

When the concentration of acetyl CoA in the mitochondria is high, citrate synthetase converts it into the form of citrate and moves it to the cytoplasm. Citric acid transported to the cytoplasm is synthesized into neutral fat through a series of processes, so if the activity of citrate synthase can be reduced, the biosynthesis of new neutral fat can be inhibited even if there is a high concentration of acetyl CoA inside the mitochondria due to excessive energy intake. there is.

The citrate synthase inhibition assay uses oxaloacetate as a reactant and analyzes the process by which citric acid synthetase produces citric acid.

The citrate synthase inhibitory effect of the modified β-MSH peptide 2 (hereinafter referred to as KKD) in Example 2 was confirmed.

Specifically, first, a KKD peptide sample was dissolved in a predetermined buffer solution at 1 to 500 μg/mL. KKD peptide, citrate synthase, and a reaction auxiliary material (acetyl CoA) were added to the buffer solution at room temperature, mixed, and allowed to stabilize for 5 minutes. Then, the reaction was started while adding a reactant (oxaloacetate). The absorbance of the sample where the reaction started was measured at 5-minute intervals for 40 minutes. Tecan Infinite 200 PRO was used as a detector, Corning 96 well plate was used as a measuring plate, and absorbance was measured at 412 nm and calculated as follows.

Formula: 100% - 100% X (absorbance _sample - absorbance _blank )/(absorbance _control - absorbance _blank )

As a result, as shown in Table 6, it was confirmed that the KKD peptide had a citrate synthase inhibitory effect. These results indicate that the KKD peptide has the effect of inhibiting the synthesis of neutral fat.

Peptide concentration (μM) 500 100 50 10 5 One inhibitory ability 18.9 1.1 0.6 1.7 0.6 0.6

Experimental example 4. In vivo Confirmation of lipolysis effect of modified peptides in

Obesity animal models were prepared to confirm the lipolytic effect of the modified β-MSH peptide 2 (hereinafter referred to as KKD) in Example 2.

Specifically, an obese animal model was prepared by feeding C57BL/6 male mice a high fat diet (HFD) for 8 weeks. After 8 weeks, it was confirmed that the HFD-fed mice gained about 1.5 times more weight than the normal feed-fed mice, and it was confirmed that the size of inguinal white adipose tissue (iWAT) was also enlarged.

After anesthetizing the manufactured obese animal, KKD peptide was applied and microcurrent was applied for 20 minutes (KKD RED), KKD peptide was applied and microcurrent was not applied (KKD Passive Diffusion), and nothing was applied (Negative Control ) was divided by

The peptide was dissolved in a phosphate buffered saline solution, soaked in a non-woven fabric, and then applied to the skin surface by pushing the mouse's abdominal hair. The group to which the microcurrent was applied was activated by attaching an electrode on a non-woven fabric and attaching an opposite electrode to the skin surface that does not come into contact with it, and then connecting it to RED.

As a result, as shown in FIGS. 4 and 5 , it was confirmed that the body weight was significantly reduced in the group to which KKD and microcurrent were applied compared to the control group.

In addition, as shown in FIGS. 6 and 7 , it was confirmed that the size and weight of inguinal white adipose tissue were reduced compared to the control group.

Then, H&E staining was performed in the following order for staining image analysis of adipose tissue. Adipose tissue was fixed in a 10% formalin solution, solidified in a paraffin solution, and the tissue sectioned at 4 μm was attached to a slide, deparaffinized in a xylene solution, and subjected to 100, 95, 90, 80, and 70% alcohol concentrations. function through. Thereafter, tissue staining was performed by washing with hematoxylin solution for 5 minutes and running water for 10 minutes, and then treating with 1% acid-alcohol for 30 seconds and eosin for 1 minute. The stained tissue was dehydrated again in the order of 70, 80, 90, 95, and 100% alcohol, and finally treated twice for 10 minutes in xylene and sealed through a transparent process.

As a result of analyzing the H&E-stained adipose tissue, it was confirmed that the size of the adipose tissue was reduced as shown in FIG. 8 .

The above results indicate that the KKD peptide, which is a modified peptide of β-MSH involved in reducing intake and regulating energy metabolism in the body, has an excellent lipolysis effect as the transdermal permeability by microcurrent increases and can be delivered systemically. can

Figure 4 is a graph showing the weight loss when the modified β-MSH peptide 2 (KKD peptide) was applied to obese mice.

Figure 5 is a photograph showing the weight loss when the modified β-MSH peptide 2 (KKD peptide) was applied to obese mice.

6 is a photograph showing that the size of inguinal white adipose tissue is reduced when modified β-MSH peptide 2 (KKD peptide) is applied to obese mice.

7 is a graph showing that the size of inguinal white adipose tissue decreased when modified β-MSH peptide 2 (KKD peptide) was applied to obese mice.

8 is a photograph showing that the size of adipose tissue is reduced when the modified β-MSH peptide 2 (KKD peptide) is applied to obese mice.

<110> BioSensor Laboratories <120> Peptides with improved transdermal drug delivery efficacy and lipolysis <130> PN210745KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> PRT <213> artificial sequence <220> <223> beta-MSH peptide <400> 1 Ala Glu Lys Lys Asp Glu Gly Pro Tyr Arg Met Glu His Phe Arg Trp 1 5 10 15 Gly Ser Pro Pro Lys Asp 20 <210> 2 <211> 20 <212> PRT <213> artificial sequence <220> <223> modified beta-MSH peptide 1 <400> 2 Lys Lys Asp Glu Gly Pro Tyr Arg Met Glu His Phe Arg Trp Gly Ser 1 5 10 15 Pro Pro Lys Asp 20 <210> 3 <211> 19 <212> PRT <213> artificial sequence <220> <223> modified beta-MSH peptide 2 <400> 3 Lys Lys Asp Glu Gly Pro Tyr Arg Met Glu His Phe Arg Trp Gly Ser 1 5 10 15 Pro Pro Lys

Claims (14)

β-melanocyte stimulating hormone (beta-Melanocyte stimulating hormone; β-MSH) peptide comprising the amino acid sequence represented by SEQ ID NO: 1, wherein the C-terminal or N-terminal amino acid sequence is modified, modified peptide. The method according to claim 1, wherein the modification of the amino acid sequence is aspartic acid (Aspartic acid), glutamic acid (Glutamic acid), lysine (Lysine), arginine (Arginine), and any one or more selected from the group consisting of histidine (Histidine) is added or deleted It is a modified peptide. The modified peptide according to claim 1, wherein the amino acid sequence is modified by deleting glutamic acid at the N-terminus and aspartic acid at the C-terminus. The modified peptide according to claim 2, wherein the N-terminal alanine is further deleted. The modified peptide according to claim 1, wherein the absolute value of the net charge of the beta-melanocyte stimulating hormone is increased to 1.5 or more by modifying the amino acid sequence. The modified peptide according to claim 1, wherein the modified peptide has higher transdermal permeability compared to before modification. The modified peptide according to claim 6, wherein the transdermal permeability is transdermal permeability due to microcurrent. The modified peptide according to claim 1, wherein the modified peptide has increased fat degradation ability compared to before modification. The modified peptide according to claim 1, wherein the modified peptide is not a cell-penetrating peptide (CPP). A cosmetic composition for lipolysis comprising the modified peptide of claim 1 as an active ingredient. A pharmaceutical composition for preventing or treating obesity comprising the modified peptide of claim 1 as an active ingredient. Health functional food for preventing or improving obesity, comprising the modified peptide of claim 1 as an active ingredient. A modified peptide comprising the step of modifying the C-terminal or N-terminal amino acid sequence of a beta-melanocyte stimulating hormone (β-MSH) peptide comprising the amino acid sequence represented by SEQ ID NO: 1 How to manufacture. The method according to claim 11, wherein the step of modifying the amino acid sequence is aspartic acid (Aspartic acid), glutamic acid (Glutamic acid), lysine (Lysine), arginine (Arginine) and histidine (Histidine) by adding any one or more selected from the group consisting of Or deletion, a method for producing a modified peptide.

Images