JP2023117892A - hepcidin binding peptide - Google Patents

Abstract

To provide a novel hepcidin inhibitor that can be readily prepared at a low production cost, has high binding specificity to hepcidin, and does not involve severe side effects.SOLUTION: The present invention provides a cyclic hepcidin binding peptide, the peptide consisting of an N-terminal peptide cyclic region, a hepcidin binding region, and a C-terminal peptide cyclic region, where, the N-terminal peptide cyclic region has a length of 0-10 amino acids, the C-terminal peptide cyclic region has a length of 0-10 amino acids, and the hepcidin binding region is an amino acid sequence represented by WDMWPSMDWKAE (SEQ ID NO: 1).SELECTED DRAWING: None

Description

The present invention relates to a peptide that specifically binds to hepcidin and its application to medicine.

Most of the iron in the body is stored in cells such as macrophages and intestinal epithelial cells. Regulation of normal blood iron levels in the body is regulated by an iron transport protein called ferroportin present in cell membranes.

When the amount of iron in the blood is low, iron in the cells moves into the blood via ferroportin. Hepcidin is secreted by the liver when blood iron levels are high. Hepcidin binds to ferroportin and induces ferroportin to intracellular lysosomes, thereby promoting ferroportin degradation. As a result, iron supply from cells to the blood is suppressed.

Today, there are reports of diseases caused by abnormalities in the above-mentioned hepcidin-ferroportin system that controls iron homeostasis, and in particular, many diseases associated with overproduction of hepcidin have been reported. In diseases associated with overproduction of hepcidin, the hepcidin concentration in the blood becomes inappropriately high, the degradation of ferroportin is accelerated more than necessary, and the supply of iron from the cells to the blood is insufficient. As a result, patients with diseases associated with overproduction of hepcidin are associated with severe anemia.

Against this background, the development of therapeutic agents for anemia that targets the functional inhibition of hepcidin is underway. For example, Patent Document 1 discloses a monoclonal antibody that specifically binds to hepcidin and a method for treating anemia and the like in mammals using the same. In addition, Patent Document 2 discloses a nucleic acid (NOX-H94) that can specifically bind to hepcidin and inhibit the hepcidin-ferroportin system. Furthermore, several reports have been made on therapeutic agents for anemia that target inhibition of hepcidin gene expression (Patent Documents 3 to 5).

U.S. Patent Application Publication No. 2008/213277 U.S. Patent Application Publication No. 2012/053234 U.S. Patent Application Publication No. 2008/260736 U.S. Patent Application Publication No. 2020/085823 U.S. Patent Application Publication No. 2016/122409

As described above, there have been several reports of anemia treatment methods that rely on inhibition of hepcidin expression or function. However, these inhibitors are (1) difficult to prepare and expensive to manufacture due to their high molecular weight, (2) insufficient specificity to hepcidin, and (3) accompanied by severe side effects. There were various problems such as Therefore, there is a strong demand for the development of new means that can solve these problems at once.

As a result of intensive studies on the above problem, the present inventors have found that a specific amino acid sequence identified by screening using a phage display method specifically binds to hepcidin despite being a relatively short sequence. In addition, the inventors found that a peptide prepared using the amino acid sequence very efficiently inhibits hepcidin's ability to promote the degradation of ferroportin. .
That is, the present invention is as follows.

[1]
A cyclic hepcidin-binding peptide, the peptide comprising:
consisting of an N-terminal peptide cyclization region, a hepcidin binding region, and a C-terminal peptide cyclization region, wherein
wherein the N-terminal peptide cyclization region has a length of 0-10 amino acids, the C-terminal peptide cyclization region has a length of 0-10 amino acids, and the hepcidin-binding region is an amino acid sequence represented by WDMWPSMDWKAE (SEQ ID NO: 1); There is a peptide.
[2]
The hepcidin-binding peptide of [1], wherein the N-terminal peptide cyclization region is 1-10 amino acids long and the C-terminal peptide cyclization region is 1-10 amino acids long.
[3]
The hepcidin-binding peptide of [1], wherein the N-terminal peptide cyclization region is 1-5 amino acids long and the C-terminal peptide cyclization region is 1-5 amino acids long.
[4]
The N-terminal peptide cyclization region comprises at least one cysteine residue and the C-terminal peptide cyclization region comprises at least one cysteine residue, wherein the cysteine present in the N-terminal peptide cyclization region and the C-terminus The hepcidin-binding peptide of [2] or [3], which is cyclized by forming a disulfide bond with a cysteine present in the peptide cyclization region.
[5]
The cysteine involved in disulfide bond formation in the N-terminal peptide cyclization region is absent at the N-terminus of the N-terminal peptide cyclization region and the cysteine involved in disulfide bond formation in the C-terminal peptide cyclization region is not present at the C-terminus of the C-terminal peptide cyclization region.
[6]
The description of [5], wherein the amino group of the amino acid present at the N-terminus of the N-terminal peptide cyclization region is acetylated, and the carboxyl group of the amino acid present at the C-terminus of the C-terminal peptide cyclization region is amidated. hepcidin-binding peptides.
[7]
A pharmaceutical composition comprising the hepcidin-binding peptide of any one of [1] to [6].
[8]
The pharmaceutical composition of [7], which is for treatment or prevention of a disease associated with overproduction of hepcidin.
[9]
Diseases associated with hepcidin overproduction are associated with infectious anemia, chronic inflammatory anemia, iron-refractory iron deficiency anemia, renal anemia, cancer-induced anemia, cancer chemotherapy-induced anemia, and neuroinflammatory disease The pharmaceutical composition according to [7], which is selected from the group consisting of anemia, atherosclerosis, cardiovascular disease, and pulmonary arterial hypertension.
[10]
The pharmaceutical composition of [7], which is for treatment or prevention of renal failure.
[11]
A hepcidin-neutralizing agent comprising the hepcidin-binding peptide according to any one of [1] to [6].
[12]
A ferroportin degradation inhibitor comprising the hepcidin-binding peptide according to any one of [1] to [6].

According to the present invention, diseases associated with overproduction of hepcidin can be treated or prevented. Also according to the present invention, renal failure can be treated or prevented.

FIG. 1 is a schematic diagram of a hepcidin-binding peptide of the present invention. Although the N-terminal peptide cyclization region and the C-terminal peptide cyclization region are shown to have the same length in FIG. 1, they may have different amino acid lengths. Either or both of the terminal peptide cyclization region and the C-terminal peptide cyclization region may be absent. FIG. 2 shows a schematic of the hepcidin-ferroportin system (A and B), a schematic of the mechanism of action that causes anemia in diseases associated with overproduction of hepcidin (C), and anemia ameliorated by the hepcidin-binding peptide of the present invention. FIG. 2D is a diagram (D) schematically showing the mechanism of action when the drug is administered. FIG. 3 shows the results of measuring the intermolecular interaction between the peptide of the present invention and hepcidin using bio-layer interferometry. It was confirmed that the peptide of the present invention specifically binds to hepcidin. FIG. 4 shows the pharmacological effects of administration of the hepcidin-binding peptide (HBP) of the present invention to renal anemia model mice. (A) Study schedule. Adenine sulfate (AdS) was administered to mice for 3 weeks (11 times) to induce renal failure (control mice were administered H ₂ O). HBP was administered from Day 15 to Day 21 (control was administered vehicle (PBS)). (B) Mouse hemoglobin levels (Hb). Hemoglobin levels were significantly lowered in mice in which renal failure was induced by AdS (renal anemic state). "*" indicates that there was a significant difference. (C to E) show improvement of renal anemia by administration of HBP of the present invention. Administration of HBP of the present invention significantly increased hemoglobin level (Hb), red blood cell count (RBC), and hematocrit level (HCt) in renal anemia model mice, which means improvement of anemia in model mice. do. FIG. 5 shows blood biochemical data when the hepcidin-binding peptide (HBP) of the present invention was administered to renal anemia model mice. (A) Urea nitrogen (BUN), an index of renal function. (B) Creatinine (CRE), an index of renal function. (C) Serum iron level (indicator of total amount of iron in blood). (D) Transferrin saturation (indicator of binding amount of blood iron and transferrin). (E) Serum ferritin level (indicator of in vivo storage of iron. In renal failure, iron is excessively stored, so this index increases). (F) C-reactive protein (CRP) content (indicator of inflammatory state in vivo; increased in renal failure). (G) Serum hepcidin levels (elevated in renal failure). (H) Serum erythropoietin level (increases with improvement of renal failure). FIG. 6 shows the results of an in vitro test on the amount of ferroportin of the hepcidin-binding peptide of the present invention. FIG. 7 shows the results of confirming the effect of the hepcidin-binding peptide of the present invention on iron levels in cells or in mouse serum. (A) Intracellular iron levels when the hepcidin-binding peptide of the present invention was added to human liver cancer-derived cells (HepG2) expressing ferroportin (FPN). The amount of intracellular iron is represented by the amount of ferritin, an intracellular iron storage protein. Ferritin levels were measured by ELISA. (B) shows the results of experiments performed with HepG2 using monkey kidney-derived fibroblasts (COS-1) expressing FPN-EGFP. Similar results were obtained with HepG2 and COS-1. "*" indicates that there was a significant difference. (C) A diagram showing changes in serum iron levels in mice (C57/BL6) to which the hepcidin-binding peptide of the present invention was administered. Simultaneous administration of hepcidin and the hepcidin-binding peptide of the present invention to mice inhibited the decrease in serum iron levels due to the action of hepcidin.

The present invention will be described in detail below. In the peptides described in this specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling.

1. Hepcidin-Binding Peptides The present invention is a cyclic hepcidin-binding peptide, said peptide consisting of an N-terminal peptide cyclization region, a hepcidin-binding region and a C-terminal peptide cyclization region, wherein said N-terminal peptide cyclization region a peptide (hereinafter referred to as , “the peptide of the present invention”, “the HBP of the present invention”, or simply “HBP”, etc.).

The peptide of the present invention is composed of three regions, an N-terminal peptide cyclization region, a hepcidin-binding region, and a C-terminal peptide cyclization region.

Both the N-terminal peptide cyclization region and the C-terminal peptide cyclization region may consist of 0-10 amino acids in length. The amino acid lengths of the N-terminal peptide cyclization region and the C-terminal peptide cyclization region may be the same or different. The N-terminal peptide cyclization region or the C-terminal peptide cyclization region composed of 0 amino acids means that the peptide of the present invention does not have the N-terminal peptide cyclization region or the C-terminal peptide cyclization region. means that

In one aspect, the N-terminal peptide cyclization region can be 1-10 amino acids long, preferably 1-5 amino acids long, more preferably 2-4 amino acids long. Also, the C-terminal peptide cyclization region may be 1-10 amino acids long, preferably 1-5 amino acids long, more preferably 2-4 amino acids long. In another aspect, the N-terminal peptide cyclization region and the C-terminal peptide cyclization region can be 1-10 amino acids long, preferably 1-5 amino acids long, more preferably 2-4 amino acids long. . In yet another aspect, both the N-terminal peptide cyclization region and the C-terminal peptide cyclization region can be 3 amino acids long.

The types of amino acids that constitute the N-terminal peptide cyclization region and the C-terminal peptide cyclization region are not particularly limited, and any amino acid may be used. Amino acids may be natural amino acids or non-natural amino acids.

The peptide of the present invention takes the form of a cyclic peptide in which amino acids contained in the N-terminal peptide cyclization region and amino acids contained in the C-terminal peptide cyclization region are covalently bonded.

Peptide cyclization by amino acids contained in the N-terminal peptide cyclization region and amino acids contained in the C-terminal peptide cyclization region may be in any form as long as the peptide of the present invention can form a ring. For example, the peptide of the present invention may be cyclized by an amide bond between the amino group of the N-terminal amino acid of the N-terminal peptide cyclization region and the carboxyl group of the C-terminal amino acid of the C-terminal peptide cyclization region. When the peptide of the present invention does not have the N-terminal peptide cyclization region and/or the C-terminal peptide cyclization region, the amino group of the N-terminal amino acid (W) of the hepcidin-binding region and/or the C-terminal of the hepcidin-binding region The carboxyl group of amino acid (E) may participate in cyclization. Alternatively, when cysteines (C) are present in both the N-terminal peptide cyclization region and the C-terminal peptide cyclization region, they may be cyclized by disulfide bonds via sulfhydryl groups of two cysteines. Alternatively, when an amino acid having an amino group in the side chain (lysine (K), etc.) is present in the N-terminal peptide cyclization region, the amino group and the carboxyl group of the C-terminal amino acid of the C-terminal peptide cyclization region may be cyclized by cyclization through an amide bond with Alternatively, the peptide of the present invention may be cyclized by a method known per se, such as cyclization by a thioether bond, CuAAC (copper(I)-catalyzed azide alkylene cycloaddition), or the like. In a preferred embodiment, the peptide of the present invention has cysteine (C) in both the N-terminal peptide cyclization region and the C-terminal peptide cyclization region, and is cyclized by a disulfide bond formed by the two cysteines. there is In a further preferred embodiment, the peptide of the present invention has cysteine (C) in both the N-terminal peptide cyclization region and the C-terminal peptide cyclization region, wherein the N-terminal peptide cyclization region The cysteine involved in disulfide bond formation in the N-terminal peptide cyclization region is absent at the N-terminus of the N-terminal peptide cyclization region, and the cysteine involved in disulfide bond formation in the C-terminal peptide cyclization region is absent in the C-terminal peptide cyclization region. It is absent at the C-terminus of the region.

In the peptide of the present invention, the hepcidin-binding region is composed of 12 amino acids represented by (N-terminal side) WDMWPSMDWKAE (C-terminal side) (SEQ ID NO: 1). This amino acid sequence is an amino acid sequence identified by the inventors using the phage display method. Interestingly, this 12-amino acid peptide does not bind to hepcidin when linear, but specifically binds to hepcidin when cyclized.

In the peptide of the present invention, any amino acid constituting the peptide of the present invention may be modified as long as the desired effect is exhibited. The type of modification is not particularly limited, and methods known per se can be used. For example, modifications include, but are not limited to, phosphorylation, amidation, acetylation, methylation, esterification, and the like. For example, a substituent (eg, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) on the side chain of any amino acid constituting the peptide of the present invention is a suitable protecting group (eg, formyl group, a C _1-6 acyl group such as a C _1-6 alkanoyl group such as an acetyl group, etc.). Alternatively, some or all of the amino acids constituting the peptide of the present invention may be substituted with D-form amino acids. Alternatively, some or all of the amino acids constituting the peptide of the present invention may be substituted with isotope-labeled amino acids.

The amino acid present at the N-terminus of the N-terminal peptide cyclization region and the amino acid present at the C-terminus of the C-terminal peptide cyclization region (in other words, the N-terminal and C-terminal amino acids of the peptide of the present invention) constitute the peptide ring. In embodiments that do not involve modification, it may be preferred that the amino acids at each of these ends are modified. In a preferred embodiment, in the peptide of the present invention, the amino acid present at the N-terminus of the N-terminal peptide cyclization region and the amino acid present at the C-terminus of the C-terminal peptide cyclization region are not involved in cyclization of the peptide, In addition, the amino group of the amino acid present at the N-terminus of the N-terminal peptide cyclization region is acetylated, and the carboxyl group of the amino acid present at the C-terminus of the C-terminal peptide cyclization region is amidated.

The C-terminus of the peptide of the present invention may be any of carboxyl group (--COOH), carboxylate ( ^--COO- ), amide (--CONH ₂ ), or ester (--COOR).

Here, R in the ester includes, for example, C _1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl and n-butyl; C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; C _6-12 aryl groups such as phenyl, α-naphthyl; phenyl-C _1-2 alkyl groups such as benzyl, phenethyl; C ₇ such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl _-14 aralkyl group; pivaloyloxymethyl group and the like can be used, but are not limited to these.

In one preferred embodiment, the peptide of the invention has the following amino acid sequence.

A C SW D M W PS M D W K A G C G

In the above amino acid sequences, "ACS" is the N-terminal peptide cyclization domain, "WDMWPSMDWKAEG" is the hepcidin binding domain, and "GCG" is the C-terminal peptide cyclization domain. A disulfide bond is formed at the two underlined Cs to cyclize the peptide. The amino group of A at the N-terminus is acetylated, and the carboxyl group of G at the C-terminus is amidated.

The peptide of the present invention can be produced according to a known general peptide synthesis protocol (eg, solid phase synthesis method (Fmoc method or Boc method), liquid phase synthesis method, etc.).

2. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions containing the peptides of the present invention (hereinafter sometimes referred to as "pharmaceutical compositions of the present invention").

The content of the peptide of the present invention in the pharmaceutical composition of the present invention is generally 0.001 to 100% by weight, preferably 0.05 to 99% by weight, more preferably 0.1 to 90% by weight of the total composition. %, but not limited to these.

The pharmaceutical composition of the invention may contain a pharmaceutically acceptable carrier in addition to the peptide of the invention.

Pharmaceutically acceptable carriers may be appropriately selected depending on the dosage form, and examples include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, and magnesium stearate. Lubricants, citric acid, menthol and other fragrances, sodium benzoate, sodium hydrogen sulfite and other preservatives, sodium citrate and other stabilizers, methylcellulose, polyvinylpyrrolidone and other suspending agents, surfactants and other dispersing agents, Examples include, but are not limited to, diluents such as water, saline, base waxes, and the like.

The pharmaceutical composition of the present invention can be administered to a subject orally or parenterally. Parenteral administration is preferred as peptides can be degraded in the stomach. Formulations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like. Formulations suitable for parenteral administration (e.g., subcutaneous injection, intramuscular injection, topical injection, intraperitoneal injection, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, including antioxidants. , buffers, bacteriostatic agents, tonicity agents and the like. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The formulation can be enclosed in a unit dose or multiple dose container such as an ampoule or a vial. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in such a manner that they can be dissolved or suspended in an appropriate sterile vehicle just prior to use.

The peptides of the present invention are capable of specifically binding to hepcidin and inhibiting its biological activity (ie, the activity of directing ferroportin to lysosomes and promoting its degradation). Therefore, the pharmaceutical composition of the present invention containing the peptide of the present invention as an active ingredient can be suitably used for treating or preventing diseases associated with overproduction of hepcidin.

Diseases associated with overproduction of hepcidin include, for example, anemia, more specifically infectious anemia, chronic inflammatory anemia, iron-refractory iron-deficiency anemia, renal anemia, carcinomatous anemia, Examples include, but are not limited to, cancer chemotherapy-induced anemia, anemia associated with neuroinflammatory diseases, and the like. Diseases associated with overproduction of hepcidin include sepsis, congestive heart failure, renal failure, diabetes, rheumatoid arthritis, atherosclerosis, Crohn's disease, hepatitis C, viral infections, arteriosclerosis, liver cirrhosis, hepatitis. , pancreatitis, cardiovascular disease, pulmonary artery hypertension, and the like, but are not limited to these. In one aspect, the disease associated with hepcidin overproduction is infectious anemia, chronic inflammatory anemia, iron-refractory iron deficiency anemia, renal anemia, cancer-induced anemia, cancer chemotherapy-induced anemia, neuroinflammation Anemia associated with sexually transmitted disease, atherosclerosis, cardiovascular disease, or pulmonary arterial hypertension.

In one aspect, the pharmaceutical composition of the present invention can be suitably used for treating or preventing renal failure. Renal failure treated or prevented by the pharmaceutical composition of the present invention may be chronic renal failure.

Subjects to which the pharmaceutical composition of the present invention is administered are not particularly limited as long as they are mammals that can suffer from diseases associated with overproduction of hepcidin. Examples of such mammals include rodents such as mice, pets such as dogs, livestock such as pigs, horses and cows, and primates such as humans, monkeys, orangutans and chimpanzees. In one aspect, the subject of administration can be a human.

The dosage of the pharmaceutical composition of the present invention may vary depending on the subject, administration method, dosage form, etc., but a person skilled in the art can use a method known per se to administer a therapeutically effective amount or A prophylactically effective amount can be determined.

The pharmaceutical composition of the present invention can also be used in combination with existing therapeutic or preventive agents for diseases associated with hepcidin overproduction.

Moreover, the term "composition" in this specification can be replaced with "agent".

In addition, "treatment" of a disease as used herein may include not only cure of the disease but also remission of the disease and amelioration of the severity of the disease.

In addition, "prevention" of a disease as used herein includes delaying the onset of the disease in addition to preventing the onset of the disease. In addition, "prevention" of a disease herein can also include preventing the recurrence of the disease after treatment or delaying the recurrence of the disease after treatment.

3. Method for treating or preventing a disease associated with overproduction of hepcidin The present invention also provides a therapeutically or preventively effective amount of the peptide of the present invention or the pharmaceutical composition of the present invention for a subject suffering from a disease associated with overproduction of hepcidin. A method for treating or preventing a disease associated with overproduction of hepcidin (hereinafter sometimes referred to as "method of the present invention") comprising administering a substance is provided.

Subjects, diseases associated with overproduction of hepcidin, doses, timing of administration, etc. in the method of the present invention are the same as those described above.

4. Hepcidin Neutralizing Agents The present invention also provides hepcidin neutralizing agents (hereinafter sometimes referred to as "neutralizing agents of the present invention") comprising the peptides of the present invention.

The peptides of the invention can specifically bind to hepcidin and inhibit its biological activity. Thus, the peptides of the invention can be used as neutralizing agents for hepcidin.

The neutralizing agent of the present invention contains the peptide of the present invention as an active ingredient. The content of the peptide of the present invention in the neutralizing agent of the present invention is usually 0.001 to 100% by weight, preferably 0.05 to 99% by weight, more preferably 0.1 to 90% by weight of the total agent. There are, but not limited to:

The neutralizing agent of the invention may contain components other than the peptide of the invention. Such components may be, for example, the pharmaceutically acceptable carriers described above.

By the way, in vivo, hepcidin binds to ferroportin and induces ferroportin to intracellular lysosomes, thereby promoting degradation of ferroportin. Since the neutralizing agent of the present invention can inhibit the ferroportin degradation promoting action of hepcidin, the neutralizing agent of the present invention can also be used as a "ferroportin degradation inhibitor" in one aspect.

The present invention will be described in more detail in the following examples, but the present invention is not limited by these examples.

[Reference Example 1] Screening for hepcidin-binding peptides using phage display method In order to identify novel peptide sequences that specifically bind to hepcidin, screening was performed by the phage display method. For phage display methods, the Ph. A D-12 phage library (manufactured by New England Biolab) was used. The amino acid sequences of peptides that bind to hepcidin were identified by the following procedures (1) to (4).

(1) Immobilization of hepcidin Biotinylated hepcidin (manufactured by Bachem) is used as a target substance, mixed with streptavidin-agarose beads (manufactured by Thermo Scientific), and immobilized on the agarose beads by biotin-streptavidin binding. (hereinafter referred to as hepcidin-immobilized beads).

(2) Panning 1× ¹⁰ phage containing Ph. The D-12 phage library and hepcidin-immobilized beads were mixed, stirred for 60 minutes, and then washed 8 times with TBS-T buffer (0.1% Tween 20 in Tris-buffered saline (pH 8.0), same below). . By this operation, phages displaying peptides capable of binding to hepcidin bind to the hepcidin-immobilized beads, and most other phages are removed.

Next, a 0.2 mM biotin solution was added and allowed to react for 30 minutes to elute the complex of phage and hepcidin from the agarose beads, and the supernatant was used as the phage eluate.

(3) Amplification of phage Phage was amplified according to the protocol provided by New England Biolab. The phage eluate was added to an E. coli (ER2738) suspension to infect and cultured with shaking (37° C.) for 4.5 hours to amplify the phage. After amplification, the supernatant is collected by centrifugation, PEG solution (20% polyethylene glycol 6000, 2M NaCl) is added to the supernatant to precipitate only phages, and the precipitate (phages) is added to 0.2 mL of TBS buffer. It was dissolved to obtain a purified phage solution. Using this purified phage solution, the same operations of panning, amplification and purification were repeated five times. In the fifth round, panning was performed up to the step of eluting the phages to obtain a phage eluate.

(4) Amino acid sequence of peptide displayed on phage The phage eluate obtained in the fifth round of panning was added to an Escherichia coli (ER2738) suspension, infected, and plated on an agar medium to form plaques. A total of 64 plaques were analyzed for phage DNA. The phage suspension obtained from each plaque was infected with Escherichia coli (ER2738), cultured with shaking for 4.5 hours to amplify the phage, and then the phage DNA was purified with PEG solution and 1 M sodium iodide solution. The DNA base sequence of the presented peptide portion was examined. The amino acid sequence of the peptide was determined from this nucleotide sequence. Among the amino acid sequences of the peptides obtained from the analysis of the phage DNA of 64 plaques, the amino acid sequence appearing most frequently, WDMWPSMDWKAE (SEQ ID NO: 1), was shown to bind specifically to hepcidin. .

[Reference Example 2] Preparation of hepcidin-binding peptide Alanine (A), cysteine (C), and serine (S) were added to the N-terminal side of the 12 amino acids obtained in Reference Example 1, and glycine (G ), cysteine (C), and glycine (G), a peptide consisting of 18 amino acids was prepared.
A C SW D M W PS M D W K A G C G

In the above amino acid sequence, a disulfide bond is formed at the two underlined C's to cyclize the peptide. In addition, the amino group of A at the N-terminus is acetylated, and the carboxyl group of G at the C-terminus is amidated. The production of this peptide was entrusted to Scrum Co., Ltd. Peptides were synthesized by Fmoc solid phase synthesis and purified by high performance liquid chromatography. In order to confirm that the prepared peptides bind to hepcidin, intermolecular interaction measurements were performed by Bio-Layer Interferometry (BLI) using an Octet K2 device from Pall Fortebio. After adsorbing biotinylated hepcidin to a streptavidin sensor chip (Pall Fortebio), the sensor chip is immersed in a solution of the peptide diluted to 2.5, 4, 6, 8 or 10 μM to measure the intermolecular interaction. did. As a result, it was confirmed that the peptide specifically binds to hepcidin (Fig. 3). The dissociation constant for this binding was 2.9×10 ⁻⁸ M. The following tests were carried out using such peptides.

[Example 1] Effects of the hepcidin-binding peptide (HBP) of the present invention in renal anemia model mice Co., Ltd.) was administered 11 times for 3 weeks to induce renal failure. Control mice received H ₂ O instead of AdS. Hepcidin-binding peptide (HBP) was administered to both AdS-treated and control mice. HBP administration was performed from the 15th day to the 21st day after the start of AdS administration (7 times). As a control experiment for HBP administration, phosphate buffer (PBS), which is a solvent for HBP, was administered. The study schedule is shown in Figure 4A. The number of mice used is as follows. untreated group (n=7), control mouse H ₂ O administration group and PBS administration group (n=8), control mouse H ₂ O administration group and HBP administration group (n=5), renal failure mouse AdS administration group and PBS-administered group (n=7), renal failure mouse AdS-administered group and HBP-administered group (n=10).

Blood was collected from the mice on day 22 and hemoglobin (Hb), red blood cell count (RBC), hematocrit (HCt), urea nitrogen (BUN), creatinine (CRE), serum iron (Serum Iron), transferrin saturation ( TSAT), serum ferritin, C-reactive protein (CRP), serum hepcidin, and serum erythropoietin (EPO) were determined. BUN and CRE are indicators of renal function, and their values increase in renal failure. Also, TSAT is an index showing how much iron present in the blood is bound to transferrin. Serum iron levels and TSAT indicate how much iron is available in the blood, and in renal failure these values decrease. Decreased serum iron levels and TSAT cause anemia. Also, the serum ferritin level is an index that reflects the amount of iron stored in the body. Values increase in renal failure. CRP is an index that reflects the inflammatory state in vivo. Values increase in renal failure. In addition, hepcidin is excessively secreted from the liver in renal failure. As a result, renal anemia is caused. The results are shown in Figures 4B-E and Figures 5A-H.

As shown in FIG. 4B, it was confirmed that the mice in which AdS was administered to induce renal failure showed a significant decrease in hemoglobin level, leading to renal anemia. In addition, as shown in FIGS. 4C to 4E, renal anemia mice administered with the HBP (hepcidin-binding peptide) of the present invention had significantly increased hemoglobin, red blood cell count, and hematocrit values compared to controls. did. In addition, as shown in FIGS. 5A to 5H, results showing improvement in anemia and improvement in renal failure were confirmed in mice in which renal failure was induced by administration of the HBP of the present invention. The above results demonstrate that the HBP of the present invention is effective in treating or preventing diseases associated with hepcidin overproduction such as anemia and renal failure.

[Example 2] Effect of the HBP of the present invention on the amount of ferroportin In order to confirm that the HBP of the present invention inhibits the action of hepcidin that promotes the degradation of ferroportin, the following in vitro experiment was performed. Changes in the amount of intracellular ferroportin due to the addition of the HBP of the present invention were confirmed in an in vitro experimental system. Human hepcidin ( Peptide Research Institute, Inc.) and the amount of ferroportin (FPN-EGFP amount) when the HBP of the present invention was added was quantified by Western blotting using an anti-ferroportin antibody. The results are shown in FIG. The peptide (cont.) added in lane 5 of FIG. 6 is not the HBP of the present invention, but a control peptide unrelated to HBP. A control peptide does not inhibit the activity of hepcidin. In lanes 6 to 10, the amount of β-actin in each protein sample in lanes 1 to 5 was examined by Western blotting using an anti-actin antibody. indicates

FPN-EGFP was expressed in HEK293 cells (lane 2). When hepcidin was added to the above cells, the amount of ferroportin (FPN-EGFP amount) decreased due to the action of hepcidin (lane 3). When hepcidin and the HBP of the present invention were added to the above cells at the same time, HBP inhibited the activity of hepcidin, thereby suppressing the decrease in the amount of FPN-EGFP (lane 4). Addition of the control peptide at the same time as hepcidin did not suppress the decrease in the amount of FPN-EGFP (lane 5).

As shown in FIG. 6, addition of the HBP of the present invention inhibited a decrease in the amount of ferroportin in cells even when hepcidin was added.

[Example 3] Effect of the HBP of the present invention on iron levels In order to confirm that the HBP of the present invention induces changes in intracellular and extracellular iron levels through inhibition of the action of hepcidin, the following experiments were conducted. Changes in intracellular and in vivo iron levels due to the addition of the HBP of the present invention were confirmed by in vitro and in vivo experimental systems.

(in vitro)
The HBP and hepcidin of the present invention were added to human liver cancer-derived cell HepG2 endogenously expressing ferroportin (FPN), and the intracellular iron content was confirmed. Intracellular iron content was determined by measuring the amount of ferritin, an iron storage protein. The amount of ferritin was measured by ELISA. The results are shown in FIG. 7A. The control peptide was the same as that used in Example 2, and "*" in the figure indicates that there was a significant difference.
A similar experiment was also performed using monkey kidney-derived fibroblasts (COS-1) in which FPN-EGFP was expressed by introducing an expression vector encoding FPN-EGFP. The results are shown in Figure 7B. The control peptide was the same as that used in Example 2, and "*" in the figure indicates that there was a significant difference.

In both cells, the addition of hepcidin alone enhanced the degradation of ferroportin, resulting in an increase in intracellular iron content. When HBP was added simultaneously with hepcidin, the activity of hepcidin was inhibited, thus suppressing the increase in intracellular iron content. Since the control peptide did not inhibit the activity of hepcidin, the increase in intracellular iron content was not suppressed.

(in vivo)
The HBP and hepcidin of the present invention were added to mice (C57/BL6) to confirm serum iron levels. The amount of iron in the serum means the amount of iron excreted from the cells in the body of the mouse to the outside of the cells. The results are shown in Figure 7C. The control peptide was the same as that used in Example 2, and "*" in the figure indicates that there was a significant difference.

When hepcidin was administered to mice, the amount of iron in their blood decreased because it accelerated the degradation of ferroportin in the cells of the mice. When HBP was administered simultaneously with hepcidin, the activity of hepcidin was inhibited, thus suppressing the decrease in blood iron level. Since the control peptide did not inhibit the activity of hepcidin, the decrease in blood iron level was not suppressed.

Both in vitro and in vivo experimental results showed that the HBP of the present invention inhibited the action of hepcidin, thereby decreasing intracellular iron levels and increasing blood iron levels in mice.

INDUSTRIAL APPLICABILITY According to the present invention, a pharmaceutical composition capable of treating or preventing diseases associated with overproduction of hepcidin can be produced at low cost. Therefore, the present invention is extremely useful in the pharmaceutical field.

Claims (12)

A cyclic hepcidin-binding peptide, the peptide comprising:
consisting of an N-terminal peptide cyclization region, a hepcidin binding region, and a C-terminal peptide cyclization region, wherein
wherein the N-terminal peptide cyclization region has a length of 0-10 amino acids, the C-terminal peptide cyclization region has a length of 0-10 amino acids, and the hepcidin-binding region is an amino acid sequence represented by WDMWPSMDWKAE (SEQ ID NO: 1); There is a peptide. 2. The hepcidin-binding peptide of claim 1, wherein the N-terminal peptide cyclization region is 1-10 amino acids long and the C-terminal peptide cyclization region is 1-10 amino acids long. 2. The hepcidin-binding peptide of claim 1, wherein the N-terminal peptide cyclization region is 1-5 amino acids long and the C-terminal peptide cyclization region is 1-5 amino acids long. The N-terminal peptide cyclization region comprises at least one cysteine residue and the C-terminal peptide cyclization region comprises at least one cysteine residue, wherein the cysteine present in the N-terminal peptide cyclization region and the C-terminus 4. The hepcidin-binding peptide according to claim 2 or 3, which is cyclized by forming a disulfide bond with a cysteine present in the peptide cyclization region. The cysteine involved in disulfide bond formation in the N-terminal peptide cyclization region is absent at the N-terminus of the N-terminal peptide cyclization region and the cysteine involved in disulfide bond formation in the C-terminal peptide cyclization region is not at the C-terminus of the C-terminal peptide cyclization region. 6. The amino group of the amino acid present at the N-terminus of the N-terminal peptide cyclization region is acetylated, and the carboxyl group of the amino acid present at the C-terminus of the C-terminal peptide cyclization region is amidated. hepcidin-binding peptides. A pharmaceutical composition comprising the hepcidin-binding peptide of any one of claims 1-6. 8. The pharmaceutical composition according to claim 7, which is for treatment or prevention of diseases associated with overproduction of hepcidin. Diseases associated with hepcidin overproduction are associated with infectious anemia, chronic inflammatory anemia, iron-refractory iron deficiency anemia, renal anemia, cancer-induced anemia, cancer chemotherapy-induced anemia, and neuroinflammatory disease 8. The pharmaceutical composition according to claim 7, which is selected from the group consisting of anemia, atherosclerosis, cardiovascular disease, and pulmonary arterial hypertension. 8. The pharmaceutical composition according to claim 7, which is used for treating or preventing renal failure. A hepcidin-neutralizing agent comprising the hepcidin-binding peptide of any one of claims 1-6. A ferroportin degradation inhibitor comprising the hepcidin-binding peptide according to any one of claims 1 to 6.