Artificial peptide and application product of artificial peptide

Abstract

An artificial peptide 20 in which a catalytic site 30 exhibiting hydrolysis activity and a cell membrane-permeable peptide unit 40 are bonded, wherein the cell membrane-permeable peptide unit 40 has 3-5 cationic amino acids at the N-terminus, and at least 3 cationic amino acids are consecutive in the amino acid sequence of the cell membrane-permeable peptide unit 40.

Description

Artificial peptides and products using artificial peptides

[0001] This invention relates to artificial peptides and application products of artificial peptides.

[0002] One method for breaking down subcutaneous fat is to administer lipolytic enzymes subcutaneously. Therefore, there is a method of administering lipolytic enzymes subcutaneously using a microneedle patch (see Patent Document 1). The administered lipolytic enzymes diffuse subcutaneously and, upon reaching the subcutaneous fat, penetrate the fat cell membrane and enter the cells, breaking down the fat within the fat cells.

[0003] Examples of lipolytic enzymes include natural lipases and their analogues, such as artificial peptides. Patent document 2 describes an artificial peptide that has amyloid-degrading properties. Patent document 3 and non-patent document 1 show peptide sequences that improve the permeability of cell membranes such as DNA.

[0004] International Publication No. 2022 / 230464, Patent No. 6455983, Patent No. 6840914

[0005] Bioorganic & Medicinal Chemistry. 2015 Aug 1, vol.23, issue 15, p. 4911-4918

[0006] Patent Document 1 lists lipase as a lipolytic enzyme. Generally, lipase is a large molecule, making it difficult to penetrate the subcutaneous tissue and permeate fat cell membranes. Therefore, when lipase is used as a lipolytic enzyme, an efficient degradation reaction cannot be obtained.

[0007] Artificial peptides like those described in Patent Document 2 have a small molecular size, which allows for high subcutaneous penetration. However, they are not designed with cell membrane permeability in mind, and therefore cannot break down fat within adipocytes.

[0008] Patent document 3 and non-patent document 1 disclose artificial peptides that improve the permeability of cell membranes, such as DNA. According to non-patent document 1, arranging nine cationic amino acids, such as arginine, induces macropinocytosis, allowing DNA to pass through the membrane. However, this known artificial peptide is not optimized for artificial peptides that undergo hydrolysis, which is the same lipolytic reaction as lipase. Therefore, simply adding an artificial peptide that allows DNA to pass through the membrane to a known artificial peptide that undergoes hydrolysis will not enable efficient lipolysis within adipocytes.

[0009] This invention has been made in view of the above-mentioned situation, and aims to provide an artificial peptide that has excellent permeability to cell membranes and can be hydrolyzed within cells.

[0010] The artificial peptide of the present invention is an artificial peptide comprising a catalytic site exhibiting hydrolytic activity and a cell membrane-permeable peptide unit, wherein the cell membrane-permeable peptide unit has 3 to 5 cationic amino acids at its N-terminus, and the amino acid sequence of the cell membrane-permeable peptide unit has at least 3 consecutive cationic amino acids.

[0011] The applied products of the artificial peptide of the present invention are applied products of the artificial peptide containing the artificial peptide of the present invention, and include detection agents, therapeutic pharmaceutical compositions, diagnostic compositions, transdermal absorption agents, transdermal absorption systems, microneedle formulations, topical preparations, cosmetics, lipolytic agents, or lipolytic agents.

[0012] According to the present invention, it is possible to provide an artificial peptide that exhibits excellent permeability to cell membranes and can be hydrolyzed within cells.

[0013] Figure 1 shows an example of the use of a microneedle device. Figure 2 is a schematic cross-sectional view of the lipolysis device and skin tissue when the lipolysis device is in use. Figure 3 is a schematic diagram of the composition of the artificial peptide. Figure 4 shows the CD spectra of artificial peptides 1 to 5. Figure 5 shows the DLS measurement results of artificial peptide 1. Figure 6 shows the DLS measurement results of artificial peptides 2 and 3. Figure 7 is a graph showing the evaluation results of the lipolysis properties of artificial peptide 1. Figure 8 is a graph showing the evaluation results of the lipolysis properties of artificial peptides 2, 3, and 4. Figure 9 is a graph showing the evaluation results of the lipolysis properties of artificial peptides 3 and 4. Figure 10 is a graph showing the evaluation results of the lipolysis properties of artificial peptide 5.

[0014] The artificial peptides and their application products of the present invention will be described below. However, the present invention is not limited to the following configurations and can be modified and applied as appropriate without altering the essence of the invention. Furthermore, combinations of two or more of the individual preferred configurations of the present invention described below also constitute the present invention.

[0015] One example of the use of the artificial peptide of the present invention is its use as a lipolytic agent. Examples of embodiments in which the artificial peptide is used as a lipolytic agent include a microneedle device and a lipolytic device.

[0016] Figure 1 shows an example of how a microneedle device is used. The microneedle device 1 includes microneedles 5 provided on the adhesive surface 3 of the surface of a substrate 4. When the user presses the microneedles 5 against the user's skin, the microneedles 5 penetrate the skin. On the side of the microneedle device 1 opposite to the microneedles 5, there is a reactant utilization section 2, which is a device that operates using aspirated reactant. A battery can be exemplified as a device in the reactant utilization section 2.

[0017] An example of the use of microneedle devices is a lipolytic device in which a lipolytic agent is administered subcutaneously, the glycerol produced by the breakdown of fat by the lipolytic agent is aspirated as a reaction product, and electricity is generated using the aspirated glycerol.

[0018] Figure 2 is a schematic cross-sectional view of the lipolysis device and skin tissue during use. The lipolysis device, a microneedle device 1, comprises a base material 4 and microneedles 5 provided on the adhesive surface 3 of the base material 4. A reaction material usage section 2, which is a battery section, is provided on the surface of the base material 4 opposite to the microneedles 5. In the battery section, the anode (glycerol oxidation section) 6 is arranged in contact with the base material 4, and a separator 8 and a cathode 7 are stacked on top of it in that order, with the anode 6 and cathode 7 connected by an external circuit 9. The microneedle 5 is inserted into the skin, and the lipolysis agent 10 diffuses from the microneedle 5 into the skin tissue 14. The lipolysis agent 10 reaches the adipocytes 15 by diffusion and breaks down the fat. The glycerol 13 produced by the breakdown of fat reaches the anode 6 through the microneedle 5 and is oxidized by the glycerol oxidase 11 present in the anode 6. This generates electrons and protons, which move to the cathode 7 via the external circuit 9 and separator 8, respectively. At the cathode 7, oxygen is reduced by oxygen reductase 12, generating electrical energy and functioning as a battery.

[0019] <Artificial Peptide> The artificial peptide of the present invention is an artificial peptide in which a catalytic site exhibiting hydrolytic activity is bound to a cell membrane-permeable peptide unit. Figure 3 is a schematic diagram of the structure of the artificial peptide. The artificial peptide 20 is formed by the binding of a catalytic site 30 to a cell membrane-permeable peptide unit 40. The catalytic site 30 and the cell membrane-permeable peptide unit 40 are peptide units having specific amino acid sequences. The cell membrane-permeable peptide unit has 3 to 5 cationic amino acids at its N-terminus, and at least 3 cationic amino acids are consecutive in the amino acid sequence of the cell membrane-permeable peptide unit.

[0020] Artificial peptides possess cell membrane-permeable peptide units, which facilitates interaction with the cell membrane. This interaction stimulates the artificial peptide to be attracted to the cell membrane surface, allowing it to efficiently enter the cell via endocytosis.

[0021] Because the artificial peptide has a catalytic site that exhibits hydrolytic activity, the artificial peptide, once it enters a cell, can hydrolyze substances within the cell.

[0022] In this specification, hydrolytic activity refers to a reaction in which chemical bonds (such as ester bonds, amide / peptide bonds, phosphate ester bonds, and glycosidic bonds) are cleaved by the addition of water molecules. The presence or absence of hydrolytic activity is detected by evaluating whether or not this decomposition reaction occurs.

[0023] To detect the presence or absence of hydrolytic activity, for example, it is necessary to evaluate whether or not components such as triglycerides, p-nitrophenyl acetate, peptides, and p-nitrophenyl phosphate can be hydrolyzed.

[0024] Specifically, the substance whose hydrolytic activity is to be evaluated is mixed with the above-mentioned components that are to be hydrolyzed, and the hydrolysis of the above-mentioned components is evaluated. The decomposition state can be evaluated by analytical methods such as changes in absorbance or LC (liquid chromatography).

[0025] If the cells are adipocytes, the artificial peptide will readily interact with the adipocyte membrane. This interaction stimulates the artificial peptide, which is then attracted to the surface of the adipocyte membrane, allowing it to efficiently enter the adipocyte via endocytosis. If the hydrolytic activity of the artificial peptide is lipolytic activity, the artificial peptide that enters the adipocyte can break down the fat within the adipocyte. In other words, it is possible to create an artificial peptide with excellent permeability to the adipocyte membrane that can achieve lipolysis in adipocytes.

[0026] The artificial peptide preferably forms a polymer, and more preferably a dimer to a tetramer. Its size is not particularly limited, but it is preferably between 3 nm and 15 nm. Whether the artificial peptide forms a polymer and its size can be estimated by DLS (dynamic light scattering) measurement.

[0027] The structure of artificial peptides (number of cationic amino acids at the N-terminus, sequential arrangement) can be determined through structural analysis. For example, amino acid sequence information can be identified by LC-MSMS. It is also possible to identify amino acids sequentially by degrading them from the N-terminus. Regarding the binding of the catalytic site and the peptide unit, if both are detected from the same molecule, it can be concluded that the catalytic site and the peptide unit are bound together within that molecule. For example, if the substance to be evaluated is a mixture, the substance to be evaluated can be isolated by LC, membrane purification, column purification, etc., and the above structural analysis can be performed on this isolated substance. Alternatively, the above characteristics / structure can be confirmed by enzymatically degrading the substance to be evaluated.

[0028] The catalytic site is a site that possesses hydrolytic activity. Preferably, the catalytic site forms an α-helix, and preferably, it is a site that contributes to structural stabilization by forming an α-helix. By forming a structurally stable α-helix both inside and outside the cell, it can interact with the cell membrane surface as designed, and the formation of a stable catalytic site inside the cell enables the hydrolysis reaction. Whether an artificial peptide forms an α-helix can be estimated by CD (circular dichroism) measurement. As a guideline, if the spectrum has a negative pole in the range of 200 nm to 210 nm, it can be estimated that an α-helix is ​​formed. Also, if the spectrum has a negative pole around 195 nm to less than 200 nm, it can be estimated that it is in a random coil state without a fixed structure.

[0029] It is preferable that the catalytic site contains the amino acid sequence of (1) or (2). (1) SDLIX ３ ALEAKIX ２ ALLX １ X １HLLSDRW (SEQ ID NO: 1) (X １ : K or R, X ２ : L or E, X ３ : R or L) (2) DLIX ２ ALLX １ X １ HLLSDRW (SEQ ID NO: 2) (X １ : K or R, X ２ : L or E)

[0030] It is preferable that the catalytic site contains any one of the amino acid sequences of (6) to (8). (6) SDLILALEAKILALLKRHLLSDRW (SEQ ID NO: 10) (7) SDLIRALEAKILALLKRHLLSDRW (SEQ ID NO: 11) (8) DLIERALLRKHLLSDRW (SEQ ID NO: 12)

[0031] The cell membrane-permeable peptide unit preferably has a cationic amino acid that imparts cell membrane permeability. Examples of the cationic amino acid include R (arginine) and K (lysine). The cell membrane-permeable peptide unit has 3 to 5 cationic amino acids at the N-terminus. Also, in the amino acid sequence of the cell membrane-permeable peptide unit, at least 3 cationic amino acids are consecutive.

[0032] Cationic amino acids improve cell membrane permeability, but if the charge derived from the cationic amino acid is too strong, cytotoxicity will occur. By setting the number of cationic amino acids to 5 or less, cytotoxicity can be reduced. Also, by setting the number of cationic amino acids to 3 or more, cell membrane permeability can be sufficiently increased. That is, by setting the number of cationic amino acids to 3 or more and 5 or less, while maintaining cell membrane permeability, cytotoxicity can be reduced and the hydrolysis target in the cell can be hydrolyzed (decompose fat in adipocytes). The cell membrane-permeable peptide unit may contain amino acids other than cationic amino acids.

[0033] It is preferable that the peptide unit with cell membrane permeability contains any of the amino acid sequences of (3) to (5). The left side of the amino acid sequence is the N-terminus. (3) RRR (4) APKRRKSGVSK (SEQ ID NO: 3) (5) PKKKKRK V (SEQ ID NO: 4)

[0034] Among these amino acid sequences, (3) consists only of cationic amino acids, and (4) and (5) are sequences containing amino acids other than cationic amino acids.

[0035] Based on the above-mentioned preferred conditions for the catalytic site and the peptide unit with cell membrane permeability, five artificial peptides (Artificial Peptide 1) to (Artificial Peptide 5) are shown as examples of preferred artificial peptides. All of these artificial peptides have a peptide unit with cell membrane permeability at the N-terminus, and the C-terminus is an amide. (Artificial Peptide 1) RRRSDLILALEA KILLKRHLLSDRW (SEQ ID NO: 5) (Artificial Peptide 2) APKRRKSGVSKSD LIRALEA KILLKRHLLSDRW (SEQ ID NO: 6) (Artificial Peptide 3) PKKKKRK VSD LIRALEA KILLKRHLLSDRW (SEQ ID NO: 7) (Artificial Peptide 4) PKKKKRK VD LIEALLRK HLLSDRW (SEQ ID NO: 8) (Artificial Peptide 5) RRRSDLIEALLRK HLLSDRW (SEQ ID NO: 9)

[0036] The artificial peptide of the present invention is preferably used in a detection agent, a therapeutic pharmaceutical composition, a diagnostic composition, a transdermal absorbent, a transdermal absorption system, a microneedle preparation, an external preparation, a cosmetic, a lipolytic agent, or a fat metabolism promoter. These products are application products of the artificial peptide of the present invention.

[0037] Examples of the artificial peptide of the present invention are shown below. The present invention is not limited only to these examples.

[0038] (1) Synthesis of peptide The above (Artificial Peptide 1) to (Artificial Peptide 5), shown as examples of preferred artificial peptides, were synthesized by the F-moc solid-phase method using a peptide synthesizer (Pioneer, Peptide synthesis System; manufactured by Applied Biosystems).

[0039] (2) CD (Circular Dichroism) Measurement Each synthesized peptide was dissolved in Tris buffer (pH 7.3) to prepare 10 μM or 100 μM peptide solutions, and the CD was measured. A Jasco J-720 spectralometer (manufactured by JASCO Corporation) was used for the measurement, and 200 μL of the peptide solution was measured using a quartz cell with a path length of 0.1 mm or 1.0 mm. The measurement conditions were: temperature 25°C, scanning wavelength 250 nm to 190 nm, data interval 0.2 nm, scanning speed 100 nm / min, response 2 sec, bandwidth 1 nm, sensitivity 10 mdeg, and number of integrations 8. The range from 250 nm to 190 nm was measured. A spectrum with a negative pole in the range of 200 to 210 nm was judged to have an α-helix, and a spectrum with a negative pole around 197 nm was judged to be in a random coil state without a fixed structure.

[0040] Figure 4 shows the CD spectra of artificial peptides 1-5. Figure 4 shows the measurement results at a concentration of 100 μM and a temperature of 37°C. From Figure 4, it was observed that artificial peptides 1, 2, and 3 have negative extremes in the range of 200 nm to 210 nm, indicating that artificial peptides 1, 2, and 3 form α-helices. Artificial peptides 4 and 5 also have negative extremes in the range of 200 nm to 210 nm, but the degree of this is observed to be somewhat weaker, indicating that artificial peptides 4 and 5 also form α-helices, but the degree of α-helix formation is less than that of artificial peptides 1-3 (indicating a mixture of α-helix and random coil structures).

[0041] (3) DLS (Dynamic Light Scattering) Measurement Artificial peptides 1 to 3 were dissolved in Tris buffer (pH 7.3) or PBS (pH 7.0), respectively, to prepare 10 μM, 50 μM, or 100 μM peptide solutions. These solutions were filtered using a 0.45 μm filter, and 16 μL was added to a quartz cell for DLS measurement. A zetasizer Nano-S (Malvern) was used for DLS measurement.

[0042] Figure 5 shows the DLS measurement results for artificial peptide 1. Figure 5 also shows the results for two different buffers, Tris buffer (pH 7.3) or PBS (pH 7.0), and two different temperatures, 25°C and 37°C. The DLS measurement of artificial peptide 1 was performed using a 50 μM peptide solution.

[0043] Figure 6 shows the DLS measurement results for artificial peptides 2 and 3. Figure 6 also shows the results at two different temperatures, 25°C and 37°C. DLS measurements of artificial peptides 2 and 3 were performed using Tris buffer (pH 7.3) and a 100 μM peptide solution.

[0044] Based on DLS measurements, assuming that all peptides form an α-helix, the elongation length is approximately 5 nm, suggesting that they are dimers or tetramers of about 7 nm in length.

[0045] (4) Evaluation of lipolysis in adipocytes 1 The lipolysis of artificial peptide 1 was evaluated using adipocytes. Cell saturations prepared in 3T3-L1 preadipocyte difference medium (KAC) at 1 × 10⁵ cells / 0.5 mL / well or 5 × 10⁴ cells / 0.5 mL / well were seeded into 24-well cell culture plates. CO ２ Inside the incubator (37°C, 5% CO2) ２ Static culture was performed in ). The culture medium was changed every 2-3 days. After confirming that confluence was reached, the culture was continued for another 2 days. The medium was removed and washed once with DPBS (Dulbecco's Phosphate-Buffered Saline). The medium was replaced with 3T3-L1 preadipocyte difference medium (KAC) and cultured for 3 days. The medium was removed and washed once with DPBS. The medium was replaced with 3T3-L1 adipocyte medium (KAC) and cultured for 7 days. After confirming lipid accumulation, the medium was replaced with 3T3-L1 adipocyte medium containing 5, 10, 20, 40, and 80 μM of peptide. CO ２ Inside the incubator (37°C, 5% CO2) ２The culture medium was incubated for three days. The culture medium was collected, and the amount of glycerol in the medium was measured using a glycerol measurement kit (Sigma-Aldrich, MAK117) according to the procedure described in the kit.

[0046] Figure 7 is a graph showing the evaluation results of the lipolytic activity of artificial peptide 1. Artificial peptide 1 achieved maximum lipolytic activity at a 40 μM treatment. Since artificial peptide 1 has three cationic amino acids, it has low cytotoxicity, so even if the peptide concentration is increased, cytotoxicity problems are less likely to occur, and the dosage can be increased, thus increasing the amount of lipolytic activity.

[0047] (5) Evaluation of intracellular lipolysis activity 2 The lipolysis activity of artificial peptides 2, 3, and 4 was evaluated using adipocytes. Cell culture was performed in the same manner as in "Evaluation of intracellular lipolysis activity 1" above, and lipolysis was confirmed. After confirming lipolysis, each peptide was replaced with 3T3-L1 adipocyte medium containing 20 μM (artificial peptides 2 and 3) or 80 μM (artificial peptide 4). ２ Inside the incubator (37°C, 5% CO2) ２ The culture medium was incubated for three days. The culture medium was collected, and the amount of glycerol in the medium was measured using a glycerol measurement kit (Sigma-Aldrich, MAK117) according to the procedure described in the kit.

[0048] Figure 8 is a graph showing the evaluation results of the lipolytic activity of artificial peptides 2, 3, and 4. Note that the standard for the relative value of glycerol production is different from that in Figure 7 (Evaluation 1). It was confirmed that artificial peptides 2, 3, and 4 all have lipolytic activity. Artificial peptides 2, 3, and 4 are peptides containing 4, 5, and 5 cationic peptides, respectively. By appropriately designing the structural stability through α-helix formation and the site where the lipolytic reaction occurs for each peptide, the peptides can stably enter adipocytes.

[0049] (6) Evaluation of intracellular lipolysis 3 The lipolysis properties of artificial peptides 3 and 4 were evaluated using adipocytes. Cell culture was performed in the same manner as in "Evaluation of intracellular lipolysis 1" above, and lipolysis was confirmed. After confirming lipolysis, each peptide was replaced with 3T3-L1 adipocyte medium containing 5, 10, 20, 40, and 80 μM. CO ２ Inside the incubator (37°C, 5% CO2) ２ The culture medium was incubated for three days. The culture medium was collected, and the amount of glycerol in the medium was measured using a glycerol measurement kit (Sigma-Aldrich, MAK117) according to the procedure described in the kit.

[0050] Figure 9 is a graph showing the evaluation results of the lipolytic activity of artificial peptides 3 and 4. Note that the standard for the relative value of glycerol production is the same as in Figure 8 (Evaluation 2), but different from Figure 7 (Evaluation 1). Both artificial peptides 3 and 4 are peptides containing five cationic peptides, but artificial peptide 4, being a lower molecular weight peptide, causes less damage to the cell membrane and has improved cell membrane permeability, making it more suitable for high-concentration treatment compared to artificial peptide 3 and allowing for higher lipolytic activity.

[0051] (7) Evaluation of intracellular lipolysis 4 The lipolysis activity of artificial peptide 5 was evaluated using adipocytes. Cell culture was performed in the same manner as in "Evaluation of intracellular lipolysis 1" above, and lipolysis was confirmed. After confirming lipolysis, the cells were replaced with 3T3-L1 adipocyte medium containing 160 μM of artificial peptide 5. ２ Inside the incubator (37°C, 5% CO2) ２ The culture medium was incubated for three days. The medium was collected, and the amount of glycerol in the medium was measured using a glycerol measurement kit (Sigma-Aldrich, MAK117) according to the procedure described in the kit. For comparison, a peptide-free level (PBS) was also evaluated in the same manner.

[0052] Figure 10 is a graph showing the evaluation results of the lipolytic activity of artificial peptide 5. Note that the relative value standard for glycerol production differs from that in Figures 7-9 (evaluations 1-3). It was confirmed that artificial peptide 5 has lipolytic activity, and in particular, that it has lipolytic activity even at a high concentration of 160 μM.

[0053] 1. Microneedle device 2. Reactant application area (battery part) 3. Application surface 4. Substrate 5. Microneedle 6. Anode 7. Cathode 8. Separator 9. External circuit 10. Lipid-degrading agent 11. Glycerol oxidase 12. Oxygen reductase 13. Glycerol 14. Skin tissue 15. Adipocyte 20. Artificial peptide 30. Catalytic site 40. Cell membrane-permeable peptide unit

Claims

1. An artificial peptide comprising a catalytic site exhibiting hydrolytic activity and a cell membrane-permeable peptide unit, wherein the cell membrane-permeable peptide unit has 3 to 5 cationic amino acids at its N-terminus, and the amino acid sequence of the cell membrane-permeable peptide unit has at least 3 consecutive cationic amino acids.

2. The artificial peptide according to claim 1, wherein the catalytic site forms an α-helix.

3. The artificial peptide according to claim 1 or 2, wherein the artificial peptide forms a polymer.

4. The artificial peptide according to any one of claims 1 to 3, wherein the catalyst site contains the amino acid sequence of (1) or (2). (1) SDLIX ３ ALEAKIX ２ ALLX １ X １ HLLSDRW (SEQ ID NO: 1) (X １ : K or R, X ２ : L or E, X ３ : R or L) (2) DLI ２ ALLX １ X １ HLLSDRW (SEQ ID NO: 2) (X １ : K or R, X ２ : L or E) 5. The artificial peptide according to any one of claims 1 to 4, wherein the cell membrane-permeable peptide unit comprises any of the amino acid sequences (3) to (5). (3) RRR (4) APKRKSGVSK (SEQ ID NO: 3) (5) PKKKRKV (SEQ ID NO: 4) 6. The artificial peptide according to any one of claims 1 to 5, wherein the hydrolytic activity is lipolytic activity.

7. An application product of an artificial peptide comprising the artificial peptide described in any one of claims 1 to 6, wherein the application product of the artificial peptide is a detection agent, a therapeutic pharmaceutical composition, a diagnostic composition, a transdermal absorption agent, a transdermal absorption system, a microneedle formulation, a topical preparation, a cosmetic, a lipolytic agent, or a lipolytic agent.

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