Artificial peptide having lipolytic activity and use thereof

Abstract

The present invention addresses the problem of providing an artificial peptide having lipolytic activity, and a lipolytic agent and a composition for improving obesity each using said artificial peptide. Provided are a peptide comprising an amino acid sequence represented by formula (I) X4DLIX3X2ALLX1X1HLLSDRW (SEQ ID NO: 1) (where: each X1 is independently K or R; X2 is L or E; X3 is X5ALEX6KI (SEQ ID NO: 2) (where X5 is R or L, and X6 is A or L) or has no amino acid present; and X4 is S or has no amino acid present) or a salt thereof, and a lipolytic agent or a composition for improving obesity containing said peptide or a salt thereof.

Description

Artificial peptides with fat-degrading properties and their applications

[0001] The present invention relates to an artificial peptide having fat-degrading properties, as well as a lipolytic agent and a composition for improving obesity using the artificial peptide.

[0002] In recent years, changes in the social environment surrounding dietary habits, namely the Westernization of diets and lack of exercise, have led to an increase in the number of obese people in Japan. Obesity refers to a condition in which excessive fat is accumulated in the body, and it can be the root cause of numerous diseases such as diabetes, dyslipidemia, hypertension, and cardiovascular disease. When obesity is accompanied by or at high risk of such diseases, it is diagnosed as obesity syndrome, and the person becomes a target for medical treatment through weight loss. Metabolic syndrome, also known as "visceral fat syndrome," is accompanied by excessive accumulation of visceral fat and carries a high risk of causing life-threatening arteriosclerotic diseases such as myocardial infarction and cerebral infarction, making early measures to reduce visceral fat necessary.

[0003] Drugs used to improve obesity or treat obesity can be broadly classified into two types: those that suppress appetite and those that inhibit fat absorption. However, these do not directly reduce body fat.

[0004] Patent Document 1 describes a leptin-active peptide having a mutation in the D-helix region that exhibits a degrading effect on adipocytes. Patent Document 2 describes a peptide that suppresses fat accumulation and degrades already accumulated fat by suppressing the expression of genes involved in lipid synthesis and increasing the expression of genes involved in lipid degradation.

[0005] On the other hand, the present inventors have discovered artificial peptides that have amyloid-degrading properties (Patent Document 3) and artificial peptides that have peptide bond cleavage activity (Patent Document 4).

[0006] Japanese Patent Publication No. 2018-505180, Japanese Patent Publication No. 2019-533680, Japanese Patent No. 6455983, Japanese Unexamined Patent Publication No. 2022-158028

[0007] The present invention aims to provide an artificial peptide having lipolytic ability, a lipolytic agent using the artificial peptide, and a composition for improving obesity.

[0008] In order to solve the above problems, the present invention includes the following inventions. [1] A peptide consisting of the amino acid sequence represented by the following formula (I) or a salt thereof: (I) X ４ DLI X ３ X ２ ALL X １ X １ HLLSDRW (SEQ ID NO: 1) (X １ is independently K or R, X ２ is L or E, X ３ is X ５ ALE X ６ KI (SEQ ID NO: 2) (X ５ is R or L, X ６ is A or L) or no amino acid is present, X ４ is S or no amino acid is present). [2] The peptide or a salt thereof according to [1], which is a peptide consisting of the amino acid sequence of the following (1) or (2). (1) SDLILALEAKILLKRHLLSDRW (SEQ ID NO: 5) (2) SDLILALE LKILLKRHLLSDRW (SEQ ID NO: 6) [3] A peptide or a salt thereof containing a peptide having lipolytic ability and a cell-penetrating peptide linked to the N-terminal side thereof, wherein the peptide having lipolytic ability has the amino acid sequence represented by the following formula (I): (I) X ４ DLI X ３ X ２ ALL X １ X １ HLLSDRW (SEQ ID NO: 1) (X １ is independently K or R, X ２ is L or E, X ３ is X ５ ALE X ６ KI (SEQ ID NO: 2) (X ５ is R or L, X ６ is A or L) or no amino acid is present, X ４A peptide or salt thereof, wherein the amino acid sequence is either S or absent, or IAALAAAHAASDAW (SEQ ID NO: 7), provided that it has 3 to 5 cationic amino acids, and excludes cell membrane permeable peptides in which at least 3 cationic amino acids are consecutive. [4] The peptide or salt thereof according to [3], wherein the cell membrane permeable peptide is a polyarginine consisting of 6 arginines. [5] The peptide or salt thereof according to [3], wherein the peptide consists of the following amino acid sequence of (3) or (4): (3)RRRRRRRSDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 11) (4)RRRRRRRIIAALAAAHAASDAW (SEQ ID NO: 12) [6] A lipolytic agent comprising the peptide or salt thereof according to any of [1] to [5]. [7] A composition for improving obesity comprising the peptide or salt thereof according to any of [1] to [5].

[0009] The present invention provides artificial peptides that have fat-degrading properties, as well as lipolytic agents and compositions for improving obesity using said artificial peptides. According to the present invention, it is possible to provide artificial peptides that can degrade fat (lipids) in fat cells.

[0010] Figure showing the three-dimensional structures of the peptides me5fR6, LP5I, and LP5IR6 derived from structure prediction by ColabFold and the positional relationship of the catalytic sites related to lipid degradation. Graph showing the results of CD measurements of the peptides me5fR6, LP5I, and LP5IR6 under the conditions of 25 °C and 37 °C. Figure showing the spectra generated under various reaction conditions of peptide LP5IR6 with beef tallow, compared with the spectrum of glycerol. Figure showing the spectra generated under various reaction conditions of lipase with beef tallow, compared with the spectrum of glycerol. Microscopic images of cells before and after adding 100 μM of peptide me5fR6 to adipocytes and culturing. Microscopic images of cells before and after adding 40 μM of peptide LP5IR6 to adipocytes and culturing. Microscopic images of cells before and after adding 0.003 mg / mL of lipase to adipocytes and culturing. Graph showing the results of semi-quantification by Oil Red O staining for peptide me5fR6. Graph showing the results of semi-quantification by Oil Red O staining for peptide LP5IR6 and lipase. Graph showing the amount of glycerol generated when peptide me5fR6 was added to adipocytes. Graph showing the glycerol concentration generated when peptide LP5IR6 and lipase were added to adipocytes. Graph showing the amount of glycerol generated on the first day after adding to lipid molecules for peptides LP5I, LP6I, and me5 as well as lipase. Graph showing the amount of glycerol generated on the third day after adding to lipid molecules for peptides LP5I, LP6I, and me5 as well as lipase. Graph showing the amount of glycerol generated on the seventh day after adding to lipid molecules for peptides LP5I, LP6I, and me5 as well as lipase.

[0011] [Artificial Peptides] The present invention provides artificial peptides having lipid-degrading properties. "Lipid-degrading properties" refers to the ability or activity to break down lipids. Lipids are one of the components that make up lipids. Examples of artificial peptides having lipid-degrading properties include peptides consisting of an amino acid sequence represented by the following formula (I) (amino acids are shown using single-letter notation) (hereinafter also referred to as "peptide of formula (I)"). (I)X ４ DLIX ３ X ２ ALLX １ X １ HLLSDRW (Sequence ID 1) (X １ Independently, K or R, and X ２ is L or E, X ３ is, X ５ ALEX ６ KI (Sequence No. 2) (X ５ is R or L, X ６ (is A or L) or if no amino acid is present, X ４ (This is either S or the amino acid is absent). Here, "X １ "is independently K or R" means that the 10th X from the N-terminus of the amino acid sequence of the peptide in Sequence ID No. 1 above. １ and the 11th X １ This means that each of them is either K or R individually, and this wording is, for example, the 10th X １ is K and the 11th X １ This includes the case where R is true, and the reverse case.

[0012] The amino acid sequence represented by formula (I) includes the following amino acid sequences (I)-1 and (I)-2: (I)-1 SDLIX ５ ALEX ６ KIX ２ ALLX １ X １ HLLSDRW (Sequence ID 3) (I)-2 DLIX ２ ALLX １ X １ HLLSDRW (Sequence ID 4) (X １ , X ２ , X ５ and X６ (As mentioned above)

[0013] In one embodiment, the peptide of formula (I) is a peptide consisting of the following amino acid sequences: (1) SDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 5) (2) SDLILALELKILALLKRHLLSDRW (SEQ ID NO: 6)

[0014] The lipodegrading or lipid-degrading activity of the peptide of formula (I) can be confirmed, for example, by the method described in Example 4 below.

[0015] In the present invention, the artificial peptide having lipodegradability may have a cell membrane permeable peptide (also referred to as "CPP") linked to its N-terminus. Therefore, the present invention provides an artificial peptide having a structure in which a cell membrane permeable peptide is linked to the N-terminus of an artificial peptide having lipodegradability, in other words, an artificial peptide comprising a lipodegradability peptide and a cell membrane permeable peptide linked to its N-terminus (hereinafter also referred to as "artificial peptide with cell membrane permeable peptide").

[0016] In artificial peptides containing cell membrane-permeable peptides, not only the peptide of formula (I) described above, but also the peptide consisting of the amino acid sequence IALAAAAHAASDAW (SEQ ID NO: 7) can be used as the lipid-degrading peptide. These peptides retain the amino acids histidine (H), serine (S), and aspartic acid (D), which are important for catalytic activity. Due to their lipid-degrading properties, these peptides can serve as catalytic sites for degrading lipid molecules in artificial peptides containing cell membrane-permeable peptides.

[0017] Cell membrane-permeable peptides are peptides that have the function of permeating the cell membrane. Cell membrane-permeable peptides are usually peptides with 30 amino acids or less, but are not limited to this as long as they have the function of permeating the cell membrane. Cell membrane-permeable peptides can promote the entry of molecules (e.g., peptides) linked to them into the cell. The artificial peptides of the present invention become more likely to interact with the cell membrane when accompanied by cell membrane-permeable peptides. Stimulated by this interaction, the artificial peptides attracted to the cell membrane surface can efficiently enter the cell by endocytosis.

[0018] The artificial peptides of the present invention (including both artificial peptides with lipid-degrading properties and artificial peptides with cell membrane-permeable peptides) can form α-helices and adopt an α-helical structure. For example, structural prediction using ColabFold can confirm that the artificial peptides of the present invention have the ability to form α-helices. Furthermore, the secondary structure of the peptides can be confirmed, for example, by CD (circular dichroism) measurement, although the results of this measurement may vary depending on the temperature at which it is measured. By adopting an α-helical structure both inside and outside the cell, they can interact with the cell membrane surface and, by forming a catalytic site inside the cell, enable the degradation of intracellular lipids.

[0019] The artificial peptides of the present invention (including both artificial peptides with lipid-degrading properties and artificial peptides with cell membrane-permeable peptides) can form polymers (e.g., di-tetramers) in solvents such as water or physiological saline (PBS) or in vivo. Polymer formation in vivo can be confirmed, for example, by structural prediction using ColabFold. Polymer formation in solvents can be confirmed, for example, by DLS (dynamic light scattering) measurement. The artificial peptides of the present invention can form polymers to form a stable catalytic site.

[0020] Regarding cell membrane permeable peptides, various peptides are known, for example, as described in Physiol. Res. 67 (Suppl. 2): S267-S279, 2018. Examples of cell membrane permeable peptides include: cationic CPPs (e.g., polyarginine, TAT, AIP6, DVB6, IRS-tag, mini-penetratin, penetratin, R9F2C, and SV-40 antigen-derived nuclear localization sequences (NLS)), amphiphilic cationic CPPs (e.g., CADY, EB-1, hCT(9-32), PTD4, MAP, Pep-1, pVEC, SymB1, transportan, and Vp1), amphiphilic neutral CPPs (e.g., MAP17, PreS2, etc.), amphiphilic anionic CPPs (e.g., GALA and MAP12), and proline-rich CPPs (e.g., (PPR)). ｎ (n=3-6), (PRR) ｎ(n=3-6), Bac-7 and SAP), and hydrophobic CPPs (e.g., BIP, C105Y, β3-integrin, K-FGF, NF-κB, Pep-7 and β1-tail). Cationic CPPs contain cationic amino acids, such as arginine (R) and lysine (K). Amphiphilic CPPs alternate polar (hydrophilic) and nonpolar (hydrophobic) amino acids in their structure, and the resulting charge can be positive, neutral, or negative. Hydrophobic CPPs contain hydrophobic amino acids in high content, such as alanine (A), leucine (L), isoleucine (I), phenylalanine (F), tryptophan (W), methionine (M), and tyrosine (Y). The cell membrane-permeable peptide is preferably one that does not hinder the formation of an α-helix or polymer by the artificial peptide obtained by linking to an artificial peptide having lipodegradability. The cell membrane-permeable peptide is preferably polyarginine. Polyarginine is a peptide consisting of, for example, 3 to 15 arginine (R) molecules. The number of arginine molecules constituting the polyarginine is preferably 6 or more and 15 or less, more preferably 6 or more and 12 or less, and even more preferably 6 or more and 8 or less. Even more preferably, it is a polyarginine consisting of 6 arginine molecules (RRRRRR: SEQ ID NO: 8).

[0021] In one embodiment, the cell membrane permeable peptide has three to five cationic amino acids and is not a cell membrane permeable peptide in which at least three cationic amino acids are consecutive (for example, polyarginine consisting of 3 to 5 arginine(R) molecules, SV-40 antigen-derived nuclear localization sequence (NLS) (PKKKRKV: SEQ ID NO: 9), and Vp1 (APKRKSGVSK: SEQ ID NO: 10)).

[0022] In one embodiment, the cell membrane permeable peptide is a polyarginine consisting of six arginine molecules, namely RRRRRR (SEQ ID NO: 8).

[0023] In one embodiment, the artificial peptide accompanied by the cell membrane permeable peptide is a peptide consisting of the following amino acid sequences: (3) RRRRRRSDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 11) (4) RRRRRRIAALAAAAHAASDAW (SEQ ID NO: 12)

[0024] The artificial peptides of the present invention can be produced by solid-phase synthesis (Fmoc method, Boc method) or liquid-phase synthesis according to known general peptide synthesis protocols. Alternatively, they can be produced by using transformants into which an expression vector containing the DNA encoding the peptide of the present invention has been introduced, or by using an in vitro transcription and translation system.

[0025] The artificial peptide of the present invention has a carboxyl group (-COOH) at the C-terminus, and a carboxylate group (-COOH) at the C-terminus. - , amide group (-CONH ２ It may be either a ) or an ester group (-COOR). Preferably an amide group (-CONH ２ ) The R in the ester group can be, for example, a methyl group, ethyl group, n-propyl group, isopropyl group, or n-butyl group. １－６ Alkyl groups, such as cyclopentyl groups and cyclohexyl groups, are C ３－８ Cycloalkyl groups, such as phenyl groups and α-naphthyl groups, etc. ６－１２ Aryl groups, such as benzyl and phenethyl groups, are phenyl-C １－２ α-naphthyl-C such as alkyl groups or α-naphthylmethyl groups １－２ C such as alkyl groups ７－１４ In addition to aralkyl groups, other examples include pivaloyloxymethyl groups, which are commonly used as oral esters. When the peptide of the present invention has a carboxyl group or carboxylate group other than the C-terminus, peptides in which these groups are amidated or esterified are also included in the peptide of the present invention.

[0026] The amino acids constituting the artificial peptide of the present invention may have side chains modified with any substituent. The substituents are not particularly limited, but examples include fluorine atoms, chlorine atoms, cyano groups, hydroxyl groups, nitro groups, alkyl groups, cycloalkyl groups, alkoxy groups, and amino groups.

[0027] Furthermore, the artificial peptide of the present invention has a protecting group (e.g., a formyl group, acetyl, etc.) on the amino group of the N-terminal serine residue. ２－６ C such as alkanoyl groups １－６ Protected by an acyl group, etc., the N-terminus is cleaved in vivo and the resulting glutamyl group is pyroglutamine-oxidized, or the substituent on the side chain of an amino acid within the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) is protected by an appropriate protecting group (e.g., formyl group, acetyl group, etc.) ２－６ C such as alkanoyl groups １－６ This also includes those protected by acyl groups, etc.

[0028] The artificial peptides of the present invention may form salts or hydrates thereof. Pharmaceutically acceptable salts are preferred. Specifically, examples include salts with acids such as hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, and palmitic acid; salts with alkali metals or alkaline earth metals such as sodium, potassium, and calcium, or with aluminum hydroxides or carbonates; and salts with triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine, arginine, and the like. The term "peptide or its salt" includes salts of peptides (preferably pharmaceutically acceptable salts) and hydrates of peptides or peptide salts.

[0029] The artificial peptides of the present invention may contain D-amino acids or non-natural amino acids, as long as the properties of the original peptide are maintained. Furthermore, other substances may be linked to the peptides, as long as the properties of the original peptide are maintained. Examples of other substances that can be linked to the peptides include other peptides, lipids, sugars or glycans, acetyl groups, natural or synthetic polymers, etc. Additionally, the peptides of the present invention may be modified by methods such as glycan addition, side-chain oxidation, or phosphorylation, as long as the properties of the original peptide are maintained.

[0030] Due to its fat-degrading properties, the artificial peptide of the present invention can be used for fat breakdown and the resulting energy generation. The artificial peptide of the present invention can be used as an active ingredient in fat-degrading agents and obesity-improving compositions described later, and can also be applied to detection agents and diagnostic compositions. Furthermore, the artificial peptide of the present invention can be used as an ingredient in pharmaceuticals, quasi-drugs, cosmetics, and food and beverages.

[0031] [Liposuction Agent] The present invention provides a liposuction agent. This liposuction agent contains the above-described artificial peptide of the present invention (including both artificial peptides having lipolysis properties and artificial peptides containing cell membrane permeable peptides) or a salt thereof as an active ingredient.

[0032] The fat-degrading agent of the present invention can decompose fats (lipids) by reacting with oils and fats. The reaction temperature is not particularly limited, but is preferably about 10°C to about 45°C, and more preferably about 25°C to about 37°C. The reaction time can be appropriately selected according to the amount of fat (lipid) to be decomposed.

[0033] The lipolytic agent of the present invention can decompose fat (lipids) within fat cells when administered to fat cells. The means by which the artificial peptide having fat-degrading properties is introduced into the fat cells are not particularly limited, and examples include physical injection by patch or the like. The artificial peptide obtained by linking the fat-degrading peptide with a cell membrane-permeable peptide actively enters the fat cells upon contact with them and exerts the ability to decompose fat (lipids) within the fat cells.

[0034] The fat-degrading agent of the present invention can be used as part of the obesity-improving composition described later. The fat-degrading agent of the present invention can be used as a substitute for lipase in the decomposition of fats and oils. The fat-degrading agent of the present invention can also decompose solid fats and oils (e.g., beef tallow) that cannot be decomposed by the natural fat-degrading enzyme lipase. The fat-degrading agent of the present invention can be used for targets where the decomposition of fats or lipids is considered beneficial, and can be applied in fields such as food and beverages, pharmaceuticals, cosmetics, industrial products, and environmental treatment. By administering, applying, or adding the fat-degrading agent of the present invention in an effective amount to a target, the fats (lipids) contained in the target can be decomposed. Examples of such "targets" include, but are not limited to, fat cells in living organisms, mammals for the purpose of obesity improvement, raw materials containing beef tallow, fats and oils in food and beverages, or targets requiring the removal of oily stains or treatment of lipid components. By using the fat-degrading agent of the present invention, animal fats such as beef tallow can be decomposed to obtain useful components such as fatty acids, which can be used as raw materials for cosmetics, quasi-drugs, soaps, detergents, lubricants, and other industrial products. Furthermore, the fat-degrading agent of the present invention can contribute to improving product quality by enabling the adjustment of fatty acid composition and removal of impurities in the oil refining process. The fat-degrading agent of the present invention can be applied to biological improvement (including treatment with pharmaceuticals) utilizing fat-degrading ability, and targets for such improvement include, for example, obesity and dyslipidemia. It can also be used for cosmetic purposes such as facial contouring, localized weight loss, skin tightening, and cellulite improvement. The fat-degrading agent of the present invention can also be used in the food processing field, for example, to improve texture and reduce fat content by fat-degrading raw materials, and to improve the flavor and digestibility of processed meat products. It is also useful as an auxiliary component to aid in fat digestion and as a cooking aid to reduce greasiness and simplify post-processing. Furthermore, the fat-degrading agent of the present invention is also useful for cleaning oily stains attached to clothing, kitchen equipment, industrial equipment, etc.

[0035] [Composition for Improving Obesity] The present invention further provides a composition for improving obesity. The composition for improving obesity of the present invention contains the above-described artificial peptide (including both artificial peptides having fat-degrading properties and artificial peptides containing cell membrane-permeable peptides) or a salt thereof as an active ingredient. In this specification, the artificial peptide or a salt thereof that is the active ingredient of the composition for improving obesity is also referred to as the "active ingredient for improving obesity".

[0036] "Obesity" refers to a state of having an excessive amount of fat in the body, and "obesity improvement" refers to a reduction in body fat and / or the suppression of body fat accumulation. Normally, a body mass index (BMI: weight (kg) divided by the square of height (m)) of 25 or higher is classified as "obese," but in this invention, "obesity improvement" also includes reducing body fat for cosmetic or health purposes (so-called diet purposes) when a person is not classified as obese by BMI. "Body fat" includes body fat and visceral fat, and when referring to "body fat," it means either body fat or visceral fat, or both. "Reduction of body fat" refers to a reduction in body fat and / or the suppression of its accumulation. "Reduction of fat" includes a reduction in the amount of fat due to lipid breakdown, a reduction in fat cells, or a reduction in the size of fat cells. According to this invention, artificial peptides break down fat (lipids) in fat cells, thereby directly reducing body fat.

[0037] The obesity-improving composition of the present invention can be used in products such as pharmaceuticals, quasi-drugs, cosmetics, and food and beverages.

[0038] The obesity-improving compositions of the present invention can be formulated by conventional methods, but are not limited to these. The products or compositions according to the present invention may be in any form of oral preparation, topical preparation, or injection.

[0039] In the present invention, there are no particular limitations on the dosage form when the product is formulated. The obesity-improving composition of the present invention can be formulated, for example, into tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, injections, infusions, suppositories, ointments, creams, liquids, gels, lotions, sticks, sprays, and transdermal patches (poultices, tapes, patches, etc.).

[0040] The obesity-improving composition of the present invention can be formulated by appropriately blending an obesity-improving active ingredient with a carrier or additive acceptable for the above-mentioned product. The blending ratio of the carrier or additive should be appropriately set based on the range commonly used in the field of the above-mentioned product. There are no particular limitations on the carriers or additives that can be blended, but examples include various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, stabilizers, emulsifiers, surfactants, preservatives, antioxidants, colorants, flavoring agents, and fragrances. Furthermore, the obesity-improving composition of the present invention can be formulated by conventional methods by blending ingredients generally used as raw materials for pharmaceuticals, quasi-drugs, cosmetics, food and beverages, etc.

[0041] Examples of food and beverage forms include functional foods (health foods) such as Foods for Specified Health Uses, Foods with Function Claims, and Foods with Nutrient Function Claims, but they may also be incorporated into general food and beverages. Examples of food and beverages include supplements; food additives; packaged beverages filled in PET bottles, cans, glass bottles, etc.; and powdered beverages to be dissolved in water (hot water), milk, fruit juice, etc. Furthermore, active ingredients such as peptides can be mixed with various known components such as sweeteners, acidulants, and vitamins to provide products that suit the user's preferences, such as tablets, capsules, drinks, soft drinks, dairy products such as yogurt, seasonings, processed foods, desserts, and confectionery. The manufacturing process for these foods is not particularly limited; for example, the target food or beverage can be manufactured by adding the obesity-improving active ingredient by appropriate means during the processing of food and beverages.

[0042] The obesity-improving composition of the present invention can be used as a cosmetic or quasi-drug. Examples of such cosmetics or quasi-drugs include, but are not limited to, skincare products, lotions, cosmetic oils, emulsions, serums, creams, pack formulations, patch formulations, sheet mask formulations, all-in-one gels, foundations, body lotions, cleansers, and facial washes.

[0043] Furthermore, the obesity-improving composition of the present invention can be administered to, for example, humans and other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cattle, dogs, monkeys, etc.). The method of administration may be either oral or parenteral administration. Parenteral administration may include, but is not limited to, transdermal administration, subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, or topical administration.

[0044] The dosage, when administered orally, is generally about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg, for a person weighing about 60 kg. When administered parenterally, for example, in the case of an injectable preparation, for a person weighing about 60 kg, the dosage by intravenous injection is generally about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg, but is not limited to these values ​​and may vary depending on the formulation method, the age, sex, symptoms, and method of administration of the recipient. The total daily dose may be a single dose or divided doses.

[0045] The present invention includes the following inventions: (1) A lipolysis method comprising the step of administering, applying, or adding an effective amount of the artificial peptide or a salt of the present invention to a target to break down a target fat. (2) The artificial peptide or a salt of the present invention for use in lipolysis. (3) A lipolysis composition comprising the artificial peptide or a salt of the present invention. (4) A method for improving obesity characterized by administering an effective amount of the artificial peptide or a salt of the present invention to a mammal. (5) The artificial peptide or a salt of the present invention for use in improving obesity. (6) Use of the artificial peptide or a salt of the present invention for manufacturing a pharmaceutical for improving obesity. (7) An obesity-improving agent comprising the artificial peptide or a salt of the present invention. (8) The artificial peptide or a salt of the present invention, compositions, formulations comprising the artificial peptide or a salt of the present invention, and methods for producing the same.

[0046] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0047] [Example 1: Synthesis and structural confirmation of artificial peptides] (1) Peptide synthesis The following three types of peptides were synthesized using a peptide synthesizer (Pioneer, Peptide synthesis System; Applied Biosystems) by the F-moc solid-phase method. The amino acid sequences of the synthesized peptides are as follows, and all have an amide at the C-terminus. me5fR6: RRRRRRIAALAAAAASDAW (SEQ ID NO: 12) LP5I: SDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 5) LP5IR6: RRRRRRSDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 11)

[0048] Furthermore, RRRRRR (SEQ ID NO: 8) in me5fR6 and LP5IR6 is a cell membrane-permeable peptide sequence, while the subsequent sequences IAALAAAHAASDAW (SEQ ID NO: 7) and SDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 5) are active sites that degrade lipid molecules. The artificial peptide obtained by linking these sequences actively enters adipocytes and exhibits the ability to degrade lipids within the adipocytes.

[0049] (2) Peptide structure prediction The structure of each synthesized peptide was predicted using ColabFold.

[0050] (3) CD (Circular Dichroism) Measurement Each synthesized peptide was dissolved in phosphate-buffered saline (PBS) (pH 7.2) to prepare a 100 μM peptide solution, and the CD spectrum was measured. A Jasco J-720 spectro-polarimeter (manufactured by JASCO Corporation) was used for the measurement, and 300 μL of the peptide solution was measured using a quartz cell with a path length of 1 mm. The measurement conditions were: temperature 25°C and 37°C, scanning wavelength 250 nm to 190 nm, data interval 0.2 nm, scanning speed 100 nm / min, response 2 seconds, bandwidth 1 nm, sensitivity 10 mdeg, and number of integrations 8 times.

[0051] (4) Results Figure 1 shows the structural prediction results for the three types of peptides. Figure 1 shows the three-dimensional structures and the arrangement of catalytic sites related to lipid degradation of peptides me5fR6, LP5I, and LP5IR6 derived from structural prediction by ColabFold. Structural prediction by ColabFold shows the three-dimensional structure calculated from the peptide sequence information, and this makes it possible to predict the structures that peptides can take in vivo. As shown in Figure 1, for all three types of peptides, it was predicted that the peptides would take an α-helix structure and that the two peptide chains would interact to form a dimer.

[0052] Figure 2 shows the CD measurement results for three types of peptides. Figure 2 is a graph showing the CD measurement results for peptides me5fR6, LP5I, and LP5IR6 under conditions of 25°C and 37°C. The vertical axis of Figure 2 is molar ellipticity ([θ] × 10⁻¹⁰). 3 ) (deg cm) 2 The graph shows the molar ellipticity ( / dmol) and the x-axis shows the wavelength (nm). In Figure 2, spectra with negative molar ellipticity extremes at 208 nm and 222 nm indicate an α-helix structure, while spectra with a negative molar ellipticity extreme at 197 nm indicate a random coil structure. Peptide me5fR6 showed similar curves at both 25°C (me5fR6_25C) and 37°C (me5fR6_37C), with a negative molar ellipticity extreme at 197 nm, indicating a random coil structure. Peptide LP5I showed similar curves at both 25°C (LP5I_25C) and 37°C (LP5I_37C), with negative molar ellipticity extremes at 208 nm and 222 nm, indicating an α-helix structure. Peptide LP5IR6 showed similar curves at both 25°C (LP5IR6_25C) and 37°C (LP5IR6_37C), exhibiting negative molar ellipticity values ​​at 197 nm, 208 nm, and 222 nm, indicating a composite structure of α-helix and random coil structures. The presence of a random coil structure by CD measurement suggests that the peptide became unstable in PBS at the measurement temperature, causing its original α-helix structure to begin to break down.

[0053] From the above, it was confirmed that all three types of peptides can form α-helices and dimers.

[0054] [Example 2: Evaluation of Lipid Decomposition of Solid Fats and Oils by Artificial Peptides] (1) Reaction of Peptides and Beef Tallow (Qualitative Measurement) A lipid decomposition experiment was conducted using beef tallow (Fujifilm Wako Pure Chemical Industries). The experimental method for the experiment using beef tallow was designed with reference to the Journal of the Chemical Society of Japan. 9. 1358-1362. (1983). An appropriate amount of beef tallow was placed in a microtube, and a solution of peptide LP5IR6 or lipase AYS Amano (Fujifilm Wako Pure Chemical Industries) dissolved in PBS was added and mixed. The resulting mixture was reacted under one of the following five conditions: (1) sonication in a bath, (2) static incubation at 25°C, (3) stirring incubation at 37°C and 2000 rpm, (4) static incubation at 37°C, and (5) mixing with surfactant Triton-X 100 and static incubation at 25°C. The sonication was performed for 2 hours, and the other reactions were performed overnight. Infrared spectroscopy was performed on the solution portion after the reaction. For the assignment of infrared spectral peaks, we referred to the control sample and the Nagasaki Environmental Health Research Center Bulletin. 63. 60-65. (2017) and the Journal of Oil Chemistry. 11(11). 577-590. (1962).

[0055] (2) Reaction of peptides with beef tallow (quantitative measurement) An appropriate amount of beef tallow was placed in a microcentrifuge tube, heated in an oven at 100°C for 3 hours to remove water and obtain a liquid lipid, which was then left to stand in a refrigerator to obtain solid fat. Peptide LP5IR6 was dissolved in PBS to obtain a peptide solution of the sample (final concentration 10 μM, 200 μL). For comparison, a sample of buffer (PBS) only was also prepared. Each sample was added to the above microcentrifuge tube and incubated. Incubation was carried out under either conditions of (1) standing at 37°C or (2) stirring at 37°C and 2000 rpm.

[0056] As described above, the sample reaction solution was removed and transferred to a separate microcentrifuge tube after 8 hours, 3 days, and 7 days of incubation. For samples containing only PBS, the sample reaction solution was removed after 7 days. 800 μL of acetone was added to the sample reaction solution to precipitate and remove the peptide, and the mixture was vortex-mixed. The precipitated peptide was then removed. The microcentrifuge tube was then left to stand overnight in a 55°C oven to evaporate the water from the sample reaction solution. Next, 200 μL of water was added to the residue and vortex-mixed for approximately 15 seconds to redissolve the residue. The glycerol produced in the redissolved sample reaction solution was quantified using a glycerol detection reagent (Cayman Chemical). This evaluated the lipid-degrading ability of the peptide.

[0057] (3) Results The spectra obtained by infrared spectroscopy measurements as described in (1) above are shown in Figures 3 and 4. These figures show the spectra produced under various reaction conditions with beef tallow for the peptide LP5IR6 (Figure 3) and lipase (Figure 4), in comparison with the spectrum of glycerol. Both Figures 3 and 4 show the results from top to bottom under the following conditions: sonication, glycerol, standing at 25°C, stirring at 37°C, standing at 37°C, and mixing with surfactant. For the spectra under each reaction condition, the vertical axis represents absorbance intensity, and the horizontal axis represents wavenumber (left is long wavenumber, right is low wavenumber, 4000 cm²). -1 ~400cm -1 This shows the area.

[0058] In Figure 3, peaks (extreme values) are observed in the spectra obtained under various reaction conditions, specifically in the area enclosed by a dotted rectangle below the label "glycerol" on the right side of the figure. This indicates that peptide LP5IR6 decomposes lipid molecules and produces glycerol. In contrast, in Figure 4, the spectra obtained under various reaction conditions do not show a peak in the same location as glycerol, indicating that the lipase used was unable to decompose the lipid molecules. Thus, when peptide LP5IR6 was reacted with beef tallow, glycerol-derived peaks (the area enclosed by a dotted rectangle below the label "glycerol" in these figures) were observed under all reaction conditions, whereas no peaks thought to be glycerol-derived were observed when lipase was used. This demonstrates that peptide LP5IR6 can decompose solid fats that cannot be decomposed by natural lipolytic enzymes such as lipase.

[0059] The results obtained using the glycerol detection reagent described in (2) above are shown in Table 1 below. In Table 1, "Sample" indicates the contents of the sample (LP5IR6, PBS) and the incubation reaction conditions (standing or stirring and the duration thereof).

[0060]

[0061] Table 1 shows that, under both static and agitated conditions, the longer the reaction time between the peptide LP5IR6 and beef tallow, the greater the amount of glycerol produced, indicating an increase in lipid degradation. Furthermore, the fact that significantly more glycerol was produced in the LP5IR6 sample than in the sample with only buffer (PBS) also indicates that LP5IR6 is degrading lipids.

[0062] [Example 3: Evaluation of Lipid Degradation of Adipocytes by Artificial Peptides] (1) Materials and Methods Primary rat visceral adipocytes (Cosmo Bio) were used as the cells, Visceral Adipocyte Culture Medium Ver.1 (Cosmo Bio) was used as the culture medium, neutral buffered formalin solution, 10% (Merck KGaA) was used as the fixation reagent, and the Lipid Assay Kit (Cosmo Bio) was used as the Oil Red O staining kit. The peptides used were me5fR6 (final concentrations 100 μM and 10 μM) and LP5IR6 (final concentrations 40 μM and 10 μM), and for comparison, lipase (final concentrations 0.012 mg / mL and 0.003 mg / mL) and PBS alone (DPBS without Calcium and Magnesium (Biological Industries)) were used.

[0063] The experimental method was as follows: Primary rat visceral adipocytes were seeded in 24-well plates and pre-cultured for 6 days. At the end of the pre-culture, differentiation into adipocytes was confirmed by microscopic observation. After collecting the culture supernatant, the culture was continued by replacing it with a medium containing peptides or lipases, and the reaction was examined. The peptides or lipases were dissolved in PBS and added to the medium at a concentration of 10% by weight. For the peptide me5fR6, the reaction was observed by culturing for 2 days, while for the peptide LP5IR6, lipase, and PBS alone, the reaction was observed by culturing for 8 hours and 24 hours after addition. After the reaction, the culture supernatant was collected and stained with Oil Red O.

[0064] The evaluation methods included cell volume reduction by microscopic observation, semi-quantification by oil red O staining, glycerol quantification using a glycerol detection reagent, and component analysis by liquid chromatography-mass spectrometry (LC-MS).

[0065] (2) Results The results of microscopic observation are shown in Figures 5 to 7. These figures are microscopic images of cells before and after addition of peptide me5fR6, LP5IR6, and lipase to adipocytes and cultured (Figure 5: me5fR6 100 μM, Figure 6: LP5IR6 40 μM, and Figure 7: lipase 0.003 mg / mL). Microscopic observation was performed before the addition of the test substance (i.e., before the reaction) and after the completion of culture (i.e., after the reaction; for me5fR6, it was 2 days after addition, and for LP5IR6 and lipase, it was 24 hours after addition). When lipase was added (Figure 7), the number of adipocytes clearly increased, whereas when peptide me5fR6 or LP5IR6 was added (Figures 5 and 6), the number of adipocytes decreased or remained almost the same with no increase observed. This suggests that the peptides me5fR6 and LP5IR6 reduce or decrease adipocytes by degrading lipid molecules within them.

[0066] The results of semi-quantification by oil red O staining are shown in Figures 8 and 9. These figures are graphs showing the results of semi-quantification by oil red O staining for peptides me5fR6 and LP5IR6, and lipase. Figure 8 shows the results of the reaction when peptide me5fR6 was added, and Figure 9 shows the results of the reaction when peptide LP5IR6 and lipase were added, respectively. In these figures, the vertical axis shows the absorbance value at a given wavelength (this represents the amount of lipid droplets), and the horizontal axis shows the reaction conditions for each sample. A lower height on the vertical axis indicates that lipids are being broken down. As shown in these figures, the absorbance values ​​on the vertical axis are clearly lower for the peptide me5fR6-added sample and the LP5IR6-added sample compared to the control sample (sample with PBS only added), and the LP5IR6-added sample showed a greater decrease than the lipase-added sample. It was also shown that increasing the amount of peptide reduced the amount of lipids. These results indicate that peptides me5fR6 and LP5IR6 reduce lipid droplets by degrading lipid molecules within adipocytes.

[0067] When lipid molecules within adipocytes are broken down into fatty acids and glycerol, glycerol is released outside the cell. Therefore, the ability to break down lipids was evaluated by detecting the glycerol released outside the cell. For this purpose, the amount of glycerol contained in the culture supernatant was quantified. The results are shown in Figures 10 and 11.

[0068] Figure 10 is a graph showing the amount of glycerol produced when peptide me5fR6 was added to adipocytes. In Figure 10, the vertical axis shows the absorbance value at 540 nm, which suggests the glycerol concentration. The horizontal axis shows the sample. In Figure 10, "d6_" indicates the supernatant data collected at the end of the 6-day preliminary culture (day 6 of culture), and "d8_" indicates the supernatant data collected after a 2-day culture reaction in each treatment group (i.e., day 8 of culture). From Figure 10, it can be seen that, particularly from the data on day 8 of culture, more glycerol was produced when peptide me5fR6 was added to adipocytes compared to the control (PBS only), and that the amount of glycerol produced increased with increasing amounts of peptide me5fR6 added. It should be noted that the glycerol detected in the supernatant up to day 6 was thought to be due to dead cells during culture, naturally occurring glycerol, or glycerol used for cell preservation when seeding adipocytes.

[0069] Figure 11 is a graph showing the glycerol concentration produced when peptide LP5IR6 and lipase were added to adipocytes. In Figure 11, the vertical axis shows the glycerol concentration (mg / L), and the horizontal axis shows the sample. In Figure 11, "P2" represents the supernatant data collected after 8 hours of culture in each treatment group, and "P3" represents the supernatant data collected after 24 hours of culture in each treatment group. From Figure 11, it can be seen that when peptide LP5IR6 was added to adipocytes, more glycerol was produced compared to the control (PBS only) or when lipase was added, the amount of glycerol produced increased depending on the amount of peptide LP5IR6 added, and the amount of glycerol produced increased depending on the reaction time using peptide LP5IR6.

[0070] Furthermore, the quantitative concentrations of the generated glycerol were 2.66 mg / L for me5fR6, 5.44 mg / L for LP5IR6, and 0.94 mg / mL for lipase.

[0071] The glycerol quantification results also showed that peptides me5fR6 and LP5IR6 have superior lipolytic properties compared to the natural lipolytic enzyme lipase.

[0072] Furthermore, Table 2 shows the LC-MS analysis results of the culture supernatant collected after the reaction when LP5IR6 was added to adipocytes. Specifically, it shows the results of the culture supernatant collected after 8 hours and 24 hours of incubation for LP5IR6 addition concentrations of 40 μM and 10 μM, respectively. In Table 2, the control group shows the results when only PBS was added.

[0073]

[0074]

[0075] Table 2 shows that fatty acids and glycerol derivatives were detected in the supernatant after adding LP5IR6 to adipocytes and culturing them. This was observed only when the peptide was added. Therefore, it was confirmed that the peptide LP5IR6 degrades lipid molecules within adipocytes.

[0076] Based on the above, peptides me5fR6 and LP5IR6 successfully entered adipocytes, degraded lipids in lipid droplets, and separated them into glycerol and fatty acids.

[0077] [Example 4: Evaluation of Lipid Degradation Capabilities of Artificial Peptides] (1) As material lipid molecules, POP (1,3-palmitoyl 2-oleoylglycerol) was used, and as degrading enzymes, the following three peptides synthesized in the same manner as in Example 1 (1) (each with an amino acid sequence shown below; all have an amide at the C terminus) and natural lipase (lipase derived from wheat germ, Sigma-Aldrich) were used. LP5I: SDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 5) LP6I: SDLILALELKILALLKRHLLSDRW (SEQ ID NO: 6) me5: SAALEAKIAALERKIIAALALAAAAHAASDAW (SEQ ID NO: 13)

[0078] (2) Experimental Method POP was mixed with one of the various peptides and lipases in 20 mM Tris-HCl buffer. At that time, POP was added to the Tris-HCl buffer at a concentration of 250 μM, peptides LP5I and LP6I at either 100 μM or 10 μM, me5 at 100 μM, and lipase at 0.22 mg / mL. Water (MQ) was used as a control sample. The mixture was incubated at 37°C for 1 day, and 200 μL of the sample was taken and mixed with 13 μL of glycerol enzyme mixture (Cayman chemical). This mixture was incubated at 25°C for 15 minutes, and the absorbance in the range of 700 to 400 nm was measured to quantify the concentration of the generated glycerol.

[0079] (3) Results The results are shown in Figures 12 to 14. These figures are graphs showing the amount of glycerol produced on day 1, day 3, and day 7 after addition of peptides LP5I, LP6I, and me5, as well as lipase, to lipid molecules (Figure 12: day 1, Figure 13: day 3, and Figure 14: day 7). In these figures, the vertical axis shows the glycerol production concentration (μM), and the horizontal axis shows the sample. From these results, it was found that peptides LP5I and LP6I have lipid-degrading properties. In particular, with the addition of LP6I at 10 μM, the glycerol production concentration increased significantly on day 3 and day 7.

[0080] It should be noted that the present invention is not limited to the embodiments and examples described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included within the technical scope of the present invention. Furthermore, all academic and patent documents mentioned herein are incorporated herein by reference.

Claims

1. A peptide consisting of the amino acid sequence represented by the following formula (I) or a salt thereof: (I) X ４ DLIX ３ X ２ ALLX １ X １ HLLSDRW (SEQ ID NO: 1) (X １ is independently K or R, and X ２ is L or E, and X ３ is X ５ ALEX ６ KI (SEQ ID NO: 2) (X ５ is R or L, and X ６ is A or L) or there is no amino acid, and X ４ is S or there is no amino acid).

2. The peptide according to claim 1 or a salt thereof, which is a peptide consisting of the following amino acid sequence (1) or (2): (1) SDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 5) (2) SDLILALELKILALLKRHLLSDRW (SEQ ID NO: 6) 3. A peptide or salt thereof comprising a lipid-degrading peptide and a cell membrane-permeable peptide linked to its N-terminus, wherein the lipid-degrading peptide has an amino acid sequence represented by the following formula (I): (I)X ４ DLIX ３ X ２ ALLX １ X １ HLLSDRW (Sequence ID 1) (X １ Independently, K or R, and X ２ is L or E, X ３ is, X ５ ALEX ６ KI (Sequence No. 2) (X ５ is R or L, X ６ (is A or L) or if no amino acid is present, X ４ A peptide consisting of the amino acid sequence IALAAAAHAASDAW (Sequence ID 7), wherein the peptide has 3 to 5 cationic amino acids, and excludes cell membrane permeable peptides in which at least 3 cationic amino acids are consecutive.

4. The peptide or salt thereof according to claim 3, wherein the cell membrane permeable peptide is a polyarginine consisting of six arginine molecules.

5. The peptide according to claim 3 or a salt thereof, which is a peptide consisting of the following amino acid sequence (3) or (4): (3) RRRRRRSDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 11) (4) RRRRRRIAALAAAAHAASDAW (SEQ ID NO: 12) 6. A lipolytic agent comprising the peptide or a salt thereof according to any one of claims 1 to 5.

7. A composition for improving obesity comprising the peptide or a salt thereof according to any one of claims 1 to 5.

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