Macrophage foam cell transformation inhibitor

Abstract

To provide novel macrophage foam cellularization inhibitors.SOLUTION: Provided is a macrophage foam cellularization inhibitor containing the peptide according to any one of the followings (a) to (c) as an active ingredient. (a) A peptide consisting of an amino acid sequence represented by Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu (SEQ ID NO:1). (b) A peptide consisting of an amino acid sequence in which one or several amino acid residues are deleted, substituted, or added in peptide (a), and which has an inhibitory action on macrophage foam cellularization. (c) A peptide having an amino acid sequence identity of 90% or more with that of peptide (a) and having an inhibitory action on macrophage foam cellularization.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to an agent for inhibiting macrophage foam cell formation. [Background technology]

[0002] In recent years, it has become common knowledge that chronic inflammation is at the root of various lifestyle-related diseases. The response from inflammation onset to resolution involves a regulatory mechanism centered on macrophages, and the mechanism by which abnormalities in macrophages are brought about in the progression of lifestyle-related diseases accompanied by chronic inflammation has attracted attention.

[0003] Atherosclerosis is a lifestyle-related disease in which the accumulation of abnormal macrophages plays a central role in the pathogenesis of the disease. Atherosclerosis is a chronic inflammatory disease of the arteries characterized by localized thickening of the interior of the arterial wall, which is caused by the formation and accumulation of foam cells.

[0004] Foam cells are macrophages with excessive lipid accumulation in their cytoplasm, and when foam cells accumulate at specific locations inside the arterial wall, plaque is formed, causing narrowing or blockage of the blood vessel. When some kind of stimulus is applied to this plaque and it ruptures, a blood clot is formed, leading to the progression of the arteriosclerosis pathology.

[0005] For the purpose of inhibiting the formation of macrophages into foam cells, for example, Patent Document 1 proposes an agent for inhibiting the formation of foam cells in macrophages in arteriosclerotic tissues, which contains ε-viniferin as an active ingredient, and Patent Document 2 proposes an agent for preventing and treating arteriosclerosis, which contains tomatidine as an active ingredient.

[0006] To further improve diseases caused by macrophage foam cell transformation, it is desirable to develop a novel agent that inhibits macrophage foam cell transformation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2012-97012 A [Patent Document 2] JP 2009-209099 A Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been proposed in view of the above, and has an object to provide a novel agent for inhibiting macrophage foam cell formation. [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above problems, the present inventors discovered that a peptide consisting of a specific amino acid sequence inhibits macrophage foam cell formation, and thus completed the present invention. More specifically, the present invention provides the following.

[0010] <1> 1. An inhibitor of macrophage foam cell formation, comprising as an active ingredient any one of the following peptides (a) to (c): (a) A peptide consisting of the amino acid sequence represented by Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu (sequence number 1). (b) A peptide having an amino acid sequence in which one or several amino acid residues in peptide (a) have been deleted, substituted or added, and having an inhibitory effect on macrophage foam cell formation. (c) a peptide having an amino acid sequence identity of 90% or more with peptide (a) and having an inhibitory effect on macrophage foam cell formation.

[0011] <2> <1> 2. A preventive or therapeutic drug for a disease for which inhibition of macrophage foam cell formation is effective, comprising the macrophage foam cell formation inhibitor described in 1. above as an active ingredient.

[0012] <3> The disease for which inhibition of macrophage foam cell formation is effective is an atherosclerosis-related disease. <2> The preventive or therapeutic agent described in the above. Effect of the Invention

[0013] According to the present invention, a novel inhibitor of macrophage foam cell formation can be provided. [Brief description of the drawings]

[0014] [Figure 1A] FIG. 2 is a staining diagram showing the results of confirming lipid accumulation in macrophages in Test Example 1. [Figure 1B] 1 is a graph showing the results of confirming lipid accumulation in macrophages in Test Example 1. [Figure 1C] FIG. 2 is a stained diagram showing the results of confirming lipid accumulation in macrophages when a p38 inhibitor according to Test Example 1 was added. [Diagram 2] 1 is a graph showing the results of confirming the phagocytic activity of macrophages in Test Example 2. [Diagram 3] FIG. 13 shows the results of confirming activation of the MAPK family in Test Example 3. [Figure 4] FIG. 13 shows the results of confirming the mRNA expression levels of representative inflammatory mediators in Test Example 4. [Figure 5A] FIG. 13 is a diagram showing the results of confirming the mRNA expression levels of ABCA1 and ABCG1 in Test Example 5. [Figure 5B] FIG. 13 shows the results of confirming the protein expression levels of ABCA1 and ABCG1 in Test Example 5. [Figure 6] 1 is a graph showing the results of confirming the phagocytic activity of macrophages in Test Example 6. [Figure 7A] 1 is a graph showing the results of confirming the expression level of ABCA1 mRNA in Test Example 7. [Figure 7B] 1 is a graph showing the results of confirming the expression level of ABCG1 mRNA in Test Example 7. [Figure 8A]FIG. 13 is a diagram showing the results of confirming the formation of arteriosclerotic pathology when a control peptide according to Test Example 8 was added. [Figure 8B] FIG. 13 is a diagram showing the results of confirming the formation of arteriosclerotic pathology when the FNIII14 peptide of Test Example 8 was added. [Figure 8C] 1 is a graph showing the percentage of Sudan-red positive area in Test Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described, however, the present invention is not limited to the following embodiment.

[0016] In this specification, a numerical range indicated with "~" indicates a range including the numerical values ​​before and after "~" as the minimum and maximum values, respectively. In describing an amino acid sequence, the left side is the N-terminus, and amino acid residues may be represented by three-letter notation well known in the art (for example, glycine residue is represented as "Gly").

[0017] <Macrophage foam cell transformation inhibitor> The macrophage foam cell formation inhibitor according to this embodiment contains, as an active ingredient, any one of the following peptides (a) to (c) (hereinafter also referred to as specific peptides): (a) A peptide consisting of the amino acid sequence represented by Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu (sequence number 1). (b) A peptide having an amino acid sequence in which one or several amino acid residues in peptide (a) have been deleted, substituted or added, and having an inhibitory effect on macrophage foam cell formation. (c) a peptide having an amino acid sequence identity of 90% or more with peptide (a) and having an inhibitory effect on macrophage foam cell formation.

[0018] The specific peptide may be produced by chemical synthesis or genetic recombination, or a commercially available product may be used. When producing the specific peptide by chemical synthesis, the liquid phase method, solid phase method, Boc method, Fmoc method, etc. may be used alone or in combination. When producing the specific peptide by genetic recombination, for example, a gene encoding the specific peptide may be introduced into a microorganism such as Escherichia coli or yeast, a plant cell, an insect cell, an animal cell, etc., using an expression vector, and expressed.

[0019] The amino acid sequence of the above peptide (a) is derived from the fibronectin type III domain (FNIII14) that constitutes fibronectin, an extracellular matrix protein molecule.

[0020] When the specific peptide is (b), the number of amino acid residues deleted, substituted, or added is not particularly limited as long as the specific peptide has an inhibitory effect on macrophage foam cell formation. For example, the number is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2. From the viewpoints of synthesis efficiency, handling, stability, etc., the total number of amino acid residues in the peptide of (b) is preferably 30 or less, more preferably 28 or less, and even more preferably 25 or less.

[0021] When the specific peptide is (c), there is no particular limitation as long as the amino acid sequence identity with the above peptide (a) is 90% or more, and the amino acid sequence identity may be 93% or more, 95% or more, or 97% or more.

[0022] The inhibition of macrophage foam cell formation by a macrophage foam cell formation inhibitor can be observed in various ways. For example, it can be observed by the suppression, reduction, disappearance, etc. of cholesterol uptake into macrophage cells before and after the use of a macrophage foam cell formation inhibitor. It may also be observed by regulating the expression of ABCA1 and ABCG1, which play a role in converting uptaken LDL (bad cholesterol) into HDL (good cholesterol) and returning it to the circulatory system. ABCA1 and ABCG1 are transporters that play an important role in the extracellular excretion of cholesterol in macrophages.

[0023] The specific peptide contained in the macrophage foam cell formation inhibitor may be modified in various ways depending on the application. For example, a water-soluble polymer such as polyethylene glycol, a sulfate group, a water-soluble amino acid, or the like may be linked to the end of the specific peptide to improve water solubility or to polymerize it. Furthermore, each of the amino acid residues constituting the specific peptide may be in either the L-form or the D-form as long as the effects of the present invention are achieved. From the viewpoint of suppressing the decomposition of the specific peptide in the body, it is preferable that at least a part of the amino acid sequence (for example, Glu at the second position) is in the D-form.

[0024] In this way, the specific peptide acts to inhibit the formation of macrophages into foam cells. Therefore, by using the specific peptide, it is possible to produce an inhibitor of the formation of macrophages into foam cells. The inhibitor of the formation of macrophages into foam cells can be used for medical purposes and also for non-medical purposes (such as research purposes).

[0025] <Preventive or therapeutic drugs> The preventive or therapeutic drug according to this embodiment contains the macrophage foam cell formation inhibitor according to this embodiment as an active ingredient. Therefore, the preventive or therapeutic drug according to this embodiment is effective against diseases for which inhibition of macrophage foam cell formation is effective. Note that "prevention" includes not only preventing the onset of a disease, but also delaying the onset of the disease. Furthermore, "treatment" includes not only eliminating or alleviating the symptoms of a disease, but also suppressing the degree of progression of the symptoms.

[0026] An example of a disease for which inhibition of macrophage foam cell formation is effective is atherosclerosis-related disease, including coronary artery disease, myocardial infarction, angina pectoris, cerebral infarction, cerebral ischemia, arteriosclerosis obliterans, intermittent claudication, gangrene, mesenteric ischemia, transient arteritis, and renal artery stenosis.

[0027] The prophylactic or therapeutic agents can be prepared by any method employed in the pharmaceutical field or by any method with suitable modifications.

[0028] The preventive or therapeutic agent may contain components other than the macrophage foam cell formation inhibitor depending on the mode of use. Examples of components other than the macrophage foam cell formation inhibitor include media and formulation additives that are generally used in the preparation of drugs. The types of media and formulation additives are not particularly limited. Media include solid media (e.g., gelatin, lactose, etc.) and liquid media (e.g., alcohol, water, physiological saline, etc.). Formulation additives include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, isotonicity agents, suspending agents, emulsifiers, buffers, preservatives, antioxidants, colorants, sweeteners, etc.

[0029] The dosage form of the prophylactic or therapeutic agent is not particularly limited and can be selected according to the application. Examples include oral preparations such as tablets, capsules, granules, powders, troches, syrups, emulsions, suspensions, films, and lemonades; parenteral preparations such as injections, drops, external preparations, suppositories, pellets, nasal preparations, pulmonary preparations (inhalants), and eye drops.

[0030] The subject of application of the prophylactic or therapeutic agent is not particularly limited, and preferred examples include mammals, etc. Mammals may be either humans or non-human animals (mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.).

[0031] The dosage of a prophylactic or therapeutic agent is appropriately determined depending on the subject of administration, the administration route, the target disease, symptoms, and the like.

[0032] The prophylactic or therapeutic agent may be administered in combination with other drugs depending on the purpose of administration, etc. The type and amount of the drug to be used in combination with the prophylactic or therapeutic agent are appropriately selected based on the effect to be obtained, etc., and may be administered together with the prophylactic or therapeutic agent or separately. EXAMPLES

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0034] <Test Example 1> The TNIIIA2 domain is present in the molecular structure of tenascin-C, an extracellular matrix molecule that is specifically and transiently highly expressed in inflammation-related areas such as arteriosclerosis. In Test Example 1, the effect of TNIIIA2-stimulated macrophages on foam cell formation was confirmed.

[0035] In the following tests, Raw264.7 cells (mouse macrophage cell line) were used. Raw264.7 cells were cultured in DMEM medium containing 10% inactivated fetal bovine serum. Raw264.7 cells were cultured at 25 × 10 4 The cells were adjusted to cells / mL and seeded on a 24-well plate coated with FBS, cultured for 2 hours, and then cultured for another 2 hours in a medium that had been changed to one without FBS. The cells thus prepared were used in the test.

[0036] A peptide consisting of the amino acid sequence Arg-Ser-Thr-Asp-Leu-Pro-Gly-Leu-Lys-Ala-Ala-Thr-His-Tyr-Thr-Ile-Thr-Ile-Arg-Gly-Val-Thr-Cys (SEQ ID NO: 2) corresponding to the TNIIIA2 region was synthesized by a conventional method. In this specification, this peptide is also referred to as "TNIIIA2 peptide".

[0037] TNIIIA2 peptide (final concentration 2.5 μg / mL) and ox-LDL (oxidized LDL) (final concentration 50 μg / mL) were added to the medium of Raw264.7 cells prepared above. After incubation at 37°C for 20 hours, the cells were washed three times with PBS, fixed with 4% formamide, and stained with Oil-Red-O.

[0038] As shown in the upper part of Figure 1A and Figure 1B, red particles (lipid droplets) were observed intracellularly in TNIIIA2-stimulated macrophages, confirming that intracellular lipid accumulation, i.e., foam cell formation, was significantly enhanced.

[0039] Next, TNIIIA2 peptide and ox-LDL were added to the medium in the same manner as above, and further, Polymyxin-B (final concentration 10 μg / mL), an inhibitor of LPS (Lipopolysaccharide), was added. LPS is an agonist of TLR4, which plays an important role in stimulating the inflammatory response by tenascin-C, and is known as an activator of phagocytosis in macrophages. After incubation at 37°C for 20 hours, the cells were washed three times with PBS, fixed with 4% formamide, and then stained with Oil-Red-O.

[0040] As shown in the lower panel of Fig. 1A, it was confirmed that intracellular lipid accumulation, i.e., foam cell formation, was significantly enhanced in TNIIIA2-stimulated macrophages even when Polymyxin-B was added. This result confirmed that the foam cell formation effect of TNIIIA2 was not due to the effect of LPS mixed into the TNIIIA2 solution.

[0041] Next, TNIIIA2 peptide and ox-LDL were added to the medium in the same manner as above, and further the p38 inhibitor SB203580 (final concentration 10 μM) was added. After incubation at 37°C for 20 hours, the cells were washed three times with PBS, fixed with 4% formamide, and then stained with Oil-Red-O.

[0042] As shown in Figure 1C, when SB203580 was added, lipid accumulation in TNIIIA2-stimulated macrophages was not observed, indicating that the promotion of foam cell formation by TNIIIA2 is dependent on p38 activation.

[0043] <Test Example 2> In Test Example 2, the phagocytic activity of TNIIIA2-stimulated macrophages was confirmed.

[0044] In this test, Raw264.7 cells were used, which were prepared in the same manner as in Test Example 1, except that the container in which they were seeded was a cover glass coated with FBS. The medium contained TNIIIA2 peptide (final concentration 2.5 μg / mL), LPS (final concentration 100 ng / mL), fluorescent FITC-labeled latex beads (final concentration 50 × 10 4 beads / mL), or SB203580 (final concentration 10 μM) was added. After incubation at 37°C for 20 minutes, the cells were washed three times with PBS and then analyzed using a fluorescent microscope. The results are shown in Figure 2. In Figure 2, "vehi" indicates a sample to which SB203580 was not added. "*" indicates that the difference is statistically significant compared to a sample to which none of the TNIIIA2 peptide, LPS, or SB203580 was added. "**" indicates that the difference is statistically significant compared to a sample to which the TNIIIA2 peptide was added and to which SB203580 was not added.

[0045] As shown in FIG. 2, the addition of TNIIIA2 peptide enhanced phagocytosis, similar to the addition of LPS. This suggests that the abnormal lipid accumulation observed in Test Example 1 was caused by the intracellular uptake of lipids that exceeded the tolerance level. Furthermore, LPS-stimulated macrophages were not affected by SB203580, whereas the phagocytosis of TNIIIA2-stimulated macrophages was suppressed by the addition of SB203580. This indicates that TNIIIA2 enhances the phagocytosis of macrophages in a p38-dependent manner.

[0046] <Test Example 3> In Test Example 3, activation of the MAPK family in TNIIIA2-stimulated macrophages was confirmed.

[0047] In this test, Raw264.7 cells were used, which were prepared in the same manner as in Test Example 1, except that the container in which the cells were seeded was a 6-well plate coated with FBS. TNIIIA2 peptide (final concentration 2.5 μg / mL) was added to the medium, and the cells were incubated at 37°C for 0.5, 1, 3, 5, 8, and 20 hours. After washing the cells three times with PBS, the cells were lysed with a lysis buffer (62.5 mM Tris, 2% SDS, 10% Glycerol, 0.005% BPB, 5% 2-ME) containing a protease inhibitor cocktail, and a certain amount of protein was separated by SDS-PAGE. Then, analysis was performed by Western blotting using anti-p-ERK antibody (Cell Signaling), anti-p-p38 antibody (Cell Signaling), and anti-actin antibody (Sigma) as primary antibodies, and HRP-labeled anti-rabbit IgG antibody (Cell Signaling) as secondary antibody. The results are shown in Figure 3.

[0048] As shown in Figure 3, TNIIIA2 stimulation was confirmed to activate p38 in macrophages. On the other hand, ERK activation was not observed. This indicates that TNIIIA2 induces p38 activation without ERK activation.

[0049] <Test Example 4> In Test Example 4, the mRNA expression levels of representative inflammatory mediators in the enhanced phagocytosis of TNIIIA2-stimulated macrophages were confirmed.

[0050] In this test, Raw264.7 cells were used, which were prepared in the same manner as in Test Example 1, except that the container in which they were seeded was a 6-well plate coated with FBS. TNIIIA2 peptide (final concentration 2.5 μg / mL), LPS (final concentration 100 ng / ml), or SB203580 (final concentration 10 μM) was added to the medium. After incubation at 37° C. for 3 hours, the cells were washed three times with PBS, total RNA was extracted, and cDNA was synthesized. Then, PCR amplification was performed using the synthesized cDNA. The results are shown in FIG. 4. In addition, “vehi” in FIG. 4 indicates a sample to which SB203580 was not added. The sequences of the primers used are as follows:

[0051] [Table 1]

[0052] As shown in Figure 4, no change in mRNA expression level was observed with TNIIIA2 stimulation for any of MCP-1, KC, MIP-2, or IL-6. This indicates that the enhancement of macrophage phagocytosis by TNIIIA2 differs from inflammatory enhancement of phagocytosis and is not accompanied by the production of representative inflammatory mediators.

[0053] <Test Example 5> In Test Example 5, the mRNA expression levels and protein expression levels of ABCA1 and ABCG1 in TNIIIA2-stimulated macrophages were confirmed.

[0054] First, TNIIIA2 peptide, LPS, or SB203580 was added in the same manner as in Test Example 4, and then cDNA was synthesized and PCR amplified using the primers shown below. The primers for GAPDH were the same as those used in Test Example 4. The results are shown in Figure 5A. In Figure 5A, "vehi" indicates a sample to which SB203580 was not added.

[0055] [Table 2]

[0056] As shown in Figure 5A, the mRNA expression levels of both ABCA1 and ABCG1 were decreased by TNIIIA2 stimulation, and this decrease in mRNA expression level was shown to be restored by the addition of SB203580.

[0057] Next, TNIIIA2 peptide was added in the same manner as in Test Example 3, and after incubation for 5 or 7 hours, analysis was performed by Western blotting using anti-ABCA1 antibody (Millipore), anti-ABCG1 antibody (Santacruz), and anti-tubulin antibody (Sigma) as primary antibodies and HRP-labeled anti-mouse IgG antibody (Cell Signaling) or HRP-labeled anti-rabbit IgG antibody (Cell Signaling) as secondary antibodies. The results are shown in Figure 5B.

[0058] As shown in FIG. 5B, the protein expression levels of both ABCA1 and ABCG1 were decreased by TNIIIA2 stimulation.

[0059] These results indicate that the abnormal accumulation of lipids observed in Test Example 1 was caused not only by the cellular uptake of lipids exceeding the tolerable amount as confirmed in Test Example 2, but also by a decrease in the ability to excrete the taken-up lipids.

[0060] <Test Example 6> In Test Example 6, the effect of FNIII14 on the phagocytic activity of macrophages was confirmed.

[0061] A peptide consisting of the amino acid sequence Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu (SEQ ID NO: 1) was synthesized by a conventional method. In this specification, this peptide is also referred to as "FNIII14 peptide".

[0062] In this test, Raw264.7 cells were used, which were prepared in the same manner as in Test Example 1, except that the container in which they were seeded was a cover glass coated with FBS. The medium contained TNIIIA2 peptide (final concentration 2.5 μg / mL), FNIII14 peptide (final concentration 5 μg / mL), fluorescent FITC-labeled latex beads (final concentration 50 × 10 4 After incubation at 37°C for 20 minutes, the cells were washed three times with PBS and analyzed using a fluorescent microscope. The results are shown in Figure 6. In Figure 6, "vehi" indicates a sample to which no peptide was added.

[0063] As shown in FIG. 6, the phagocytosis of macrophages enhanced by the addition of TNIIIA2 peptide was suppressed by the addition of FNIII14 peptide.

[0064] <Test Example 7> In Test Example 7, the mRNA expression levels of ABCA1 and ABCG1 were confirmed when the FNIII14 peptide was added. In addition, since the TNIIIA2 region is known to have the ability to activate β1 integrin, the mRNA expression levels were also confirmed when other β1 integrin activation reagents were used.

[0065] In this test, Raw264.7 cells were used, which were prepared in the same manner as in Test Example 1, except that the container in which they were seeded was a 6-well plate coated with FBS. TNIIIA2 peptide (final concentration 2.5 μg / mL), FNIII14 peptide (final concentration 5 μg / mL), MgCl2 (final concentration 1 mM / L), anti-9EG7 antibody (IgG) (final concentration 30 μg / mL), or control IgG (final concentration 30 μg / mL) were added to the medium. After incubation at 37°C for 3 hours, the cells were washed three times with PBS, total RNA was extracted, and cDNA was synthesized. Then, real-time PCR amplification was performed using the synthesized cDNA with the SYBR (registered trademark) Green system. The results of ABCA1 are shown in Figure 7A, and the results of ABCG1 are shown in Figure 7B. In addition, "vehi" in Figures 7A and 7B indicates a sample to which no peptide or reagent was added. In addition, "*" indicates that the results are statistically significant compared to vehi. "**" indicates that the difference is statistically significant compared to the sample to which control IgG was added. "#" indicates that the difference is statistically significant compared to the sample to which TNIIIA2 peptide was added. The primers used were the same as those used in Test Example 5.

[0066] As shown in Figures 7A and 7B, the mRNA expression levels of ABCA1 and ABCG1, which were decreased by the addition of TNIIIA2 peptide, were restored by the addition of FNIII14 peptide. It was also confirmed that the mRNA expression levels of ABCA1 and ABCG1 were decreased by the addition of MgCl2 and anti-9EG7 antibody.

[0067] The results of Test Examples 6 and 7 demonstrated that the "enhanced phagocytosis" and "suppression of ABCA1 / ABCG1 expression", which are presumed to be the mechanisms by which TNIIIA2 induces macrophage foam cell transformation, are related to the activation of β1 integrin, and that these effects are canceled by the addition of FNIII14 peptide.

[0068] <Test Example 8> In Test Example 8, the effect of FNIII14 on the formation of arteriosclerosis pathology was confirmed using high-fat diet-fed LDLR-KO mice.

[0069] First, a peptide consisting of the amino acid sequence Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Leu-Ile-Val-Tyr-Ala-Thr-Tyr-Ile (SEQ ID NO: 17) was synthesized by a conventional method. This peptide was obtained by shuffling the amino acid sequence of the active portion of the FNIII14 peptide to lose its activity. In this specification, this peptide is also referred to as a "control peptide."

[0070] 10-week-old male LDL-R KO mice were randomly divided into three groups and started to be fed a high-fat diet. From the age of 12 weeks, FNIII14 peptide (100 μg), control peptide (100 μg), or saline was administered every other day. At the age of 20 weeks, the mice were sacrificed and the aorta was excised. Then, staining was performed using Sudan IV. The staining results when the control peptide was administered are shown in FIG. 8A, and the staining results when the FNIII14 peptide was administered are shown in FIG. 8B. The percentage of the Sudan-red positive area is shown in FIG. 8C. In FIG. 8C, "vehicle" indicates the group administered saline. In addition, "*" indicates that the results are statistically significant compared to the group administered the control peptide.

[0071] As shown in Figures 8A to 8C, the percentage of Sudan-red positive area was significantly decreased in the group administered with FNIII14 peptide compared to the groups administered with saline or the control peptide. This confirmed that FNIII14 peptide prevents the formation of atherosclerotic pathology at the animal level.

Claims

1. An inhibitor of macrophage foam cell formation, comprising as an active ingredient any one of the following peptides (a) to (c): (a) A peptide consisting of the amino acid sequence represented by Thr-Glu-Ala-Thr-Ile-Thr-Gly-Leu-Glu-Pro-Gly-Thr-Glu-Tyr-Thr-Ile-Tyr-Val-Ile-Ala-Leu (SEQ ID NO: 1). (b) A peptide having an amino acid sequence in which one or several amino acid residues in peptide (a) have been deleted, substituted, or added, and having the activity of inhibiting macrophage foam cell formation. (c) a peptide having an amino acid sequence identity of 90% or more with peptide (a) and having an inhibitory effect on macrophage foam cell formation.

2. A preventive or therapeutic drug for a disease for which inhibition of macrophage foam cell formation is effective, comprising the macrophage foam cell formation inhibitor according to claim 1 as an active ingredient.

3. The preventive or therapeutic agent according to claim 2, wherein the disease for which inhibition of macrophage foam cell formation is effective is an atherosclerosis-related disease.

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