WO2011027839A1

WO2011027839A1 - Cholesterol-efflux peptide

Info

Publication number: WO2011027839A1
Application number: PCT/JP2010/065070
Authority: WO
Inventors: 朔啓二郎; 安東勢津子; 上原吉就
Original assignee: 学校法人福岡大学
Priority date: 2009-09-02
Filing date: 2010-09-02
Publication date: 2011-03-10
Also published as: JP5742029B2; JPWO2011027839A1

Abstract

Disclosed is a peptide which has an effect of promoting cholesterol efflux and another effect of generating HDL. This cholesterol-efflux peptide has an effect of generating HDL in addition to the effect of promoting cholesterol efflux. A medicinal composition that comprises the aforesaid cholesterol-efflux peptide as the main active ingredient is useful in promoting cholesterol efflux, increasing serum HDL concentration, activating LCAT and promoting RCT and, therefore, can be used for preventing and treating cardiovascular diseases, for example, angina, hypertension, hyperlipemia such as hypercholesterolemia and arterial sclerosis such as atherosclerosis, cardiovascular diseases relating to cardiovascular disorders that are induced by medical treatments using a balloon, a stent or the like such as re-stenosis caused by atherosclerotic plaque, and disorders related thereto.

Description

Cholesterol export peptide

This invention relates to a cholesterol export peptide. More specifically, the present invention relates to a cholesterol export peptide that also has an HDL producing action.

¡Cholesterol is an essential component for constituting and maintaining a living body. However, since cholesterol is a kind of lipid, water does not dissolve in the main blood, and cannot be transported with blood as it is. Therefore, cholesterol is transported in the blood in the form of a complex called lipoprotein by binding to apolipoprotein in vivo. This lipoprotein is generally classified into four types: chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) depending on the density and specific gravity of apolipoprotein particles. Largely classified.

Among these lipoproteins, low density lipoprotein (LDL) and high density lipoprotein (HDL) are mainly involved in the transport of cholesterol in the blood. Low density lipoprotein (LDL) plays an important role in delivering cholesterol to cells. On the other hand, high density lipoprotein (HDL) plays an important role in transporting cholesterol from cells to the liver. Therefore, under normal conditions, LDL and HDL are balanced, and the delivery and removal of cholesterol is properly performed so that no problems arise. However, once the balance between LDL and HDL breaks down, it is known that cholesterol cannot be controlled in the body, causing various abnormalities, disorders, and diseases in the body.

Especially, it is well known that the high value of low density lipoprotein (LDL) cholesterol is an extremely high risk factor for cardiovascular diseases such as arteriosclerosis. In this sense, low density lipoprotein (LDL) cholesterol is commonly referred to as “bad cholesterol”. Therefore, as a first option for preventing or treating cardiovascular disease, LDL cholesterol lowering therapy for lowering LDL cholesterol is applied. This LDL cholesterol lowering therapy is generally performed by administration of a cholesterol lowering drug such as a statin. However, the current situation is that the onset of cardiovascular disease cannot be sufficiently suppressed by such LDL cholesterol lowering therapy alone (see, for example, Non-Patent Document 1).

On the other hand, as described above, high-density lipoprotein (HDL) plays an important role in the so-called cholesterol reverse transfer system that transports cholesterol from cells to the liver, that is, as a scavenger for tissue cholesterol. Plays an important role. For this reason, high density lipoprotein (HDL) cholesterol is called “good cholesterol”, whereas low density lipoprotein (LDL) cholesterol is commonly referred to as “bad cholesterol”. It is commonly called. Therefore, the blood concentration of high density lipoprotein (HDL) cholesterol is required to be higher than the prescribed concentration. On the other hand, if the HDL cholesterol is lower than the specified value, a sufficient amount of cholesterol is not transported to the liver and is not decomposed, resulting in an increase in the amount of cholesterol in the blood, resulting in cardiovascular It has been shown to be one of disease risk factors (see, for example, Non-Patent Documents 2 and 3). Therefore, HDL cholesterol enhancing action therapy is attracting attention as the next therapeutic target of LDL cholesterol lowering therapy (see, for example, Non-Patent Documents 4 and 5).

Currently, as HDL cholesterol enhancing action therapy, statins as HDL cholesterol increasing agents, drug administration therapy such as cholesterol ester transfer protein inhibitors are being carried out. However, none of these are sufficiently effective. In other words, currently available drugs for preventing and treating cholesterol do not have a satisfactory HDL cholesterol increasing effect (see Non-Patent Document 1, for example).

On the other hand, in-vivo experimental data showing that apolipoprotein AI (ApoA-I), which is HDL タンパク or its main protein component, prevents atherosclerosis symptoms have been reported. Yes. That is, in humans, serum ApoA-I (SEQ ID NO: 1) and atherogenesis are in an inversely proportional relationship (see, for example,

Non-Patent Documents

2 and 6 and Patent Document 1), and HDL components are -It has been clarified that the risk of atherosclerosis is reduced (see, for example, Non-Patent Document 7 and Patent Document 1). It has also been clarified that HDL or ApoA-I causes plaque regression (see, for example, Non-Patent Documents 8 and 9). Based on these findings, research and development have been promoted targeting therapeutic targets of apolipoprotein AI (ApoA-I) itself, which is apolipoprotein which is HDL and its main protein component.

As a result of these research and development, various synthetic HDL (reconstituted high density lipoprotein: rHDL) for enhancing HDL action have been produced (for example, see Non-Patent Document 10). As such rHDL, for example, a conventional purification means such as HDL (see, for example, Non-Patent Document 11), chromatography, etc., which is isolated by a series of ultracentrifugation treatment after treating human plasma or serum, is used. And purified apolipoprotein AI (ApoA-I) (see, for example, Patent Document 2 and Non-Patent Document 12). In addition, ApoA-I or a mutant thereof is also produced by gene recombination (see, for example,

Patent Documents

1 and 5 and Non-Patent Document 13).

The HDL or ApoA-I produced as described above has been prepared various synthetic HDL (rHDL) in combination with lipids. Examples of such rHDL include rHDL prepared from plasma, ApoA-I and soybean lecithin (Patent Document 6), and rHDL prepared from ApoA-I and phosphatidylcholine extracted from egg or soybean (Patent Document 7). And rHDL produced from ApoA-I and dimyristoylphosphatidylcholine (DMPC) (see, for example, Non-Patent Document 14). In addition to these, as rHDL produced by the present inventors, rHDL (POPC / ApoA-) produced from apolipoprotein AI (ApoA-I) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) I) (see, for example, Non-Patent Documents 1 and 15) and the above rHDL (POPC / ApoA-I), sphingosine-1-phosphate (SIP), one of the active phospholipids, is added And new rDHL (POPC / SIP / ApoA-I) (see Non-Patent Document 3, for example). In rHDL (POPC / ApoA-I) produced by the present inventors, it was confirmed that extra cholesterol accumulated in cells was extracted by co-culture with macrophages (for example, non-patented) References 1 and 3). Further, this new type of rHDL (POPC / SIP / ApoA-I) showed an action of promoting the formation of coronary endothelial cells in vitro in addition to the action of pulling out cholesterol in vitro (for example, non-patent literature) 15). In addition to these results, rHDL (ApoA-I) infusion resulted in increased plasma high-density lipoprotein anti-inflammatory action and cholesterol removal ability. It has also been shown that infusion of ApoA-I) sputum may have a potential preventive effect on atherogenesis (see, for example, Non-Patent Document 16). From the results of rHDL, rHDL has attracted attention.

However, on the other hand, it has also been highlighted that there are various problems in actually using apolipoprotein AI (ApoA-I) or HDL as a medicine. Since ApoA-I is a large molecular protein consisting of 243 amino acid residues, its production method is complicated and difficult, its production cost is expensive, and stability during storage, in vivo It has also been found that various difficult problems in production and reproducibility such as delivery and half-life of the active substance must be solved (see, for example, Patent Document 1).

On the other hand, various peptides that mimic the structure or activity of ApoA-I have been produced on the strong ground that ApoA-I itself has a cholesterol export function. In designing such pseudopeptides, it has been reported that the key activity of ApoA-I depends on the presence of a large number of repeats of the unique secondary structural properties of this protein, namely the class A amphipathic α-helix ( For example, see Non-Patent Document 17), the focus has been on forming a class A amphipathic α-helix.

Such ApoA-I pseudopeptides include, for example, a 198-2192 fragment of ApoA-I consisting of 22 residues in which only Glu, Lys and Leu are regularly arranged so as to form an amphipathic α-helix. Peptide having% sequence homology (ELK peptide) (see, for example, Non-patent Documents 18 and 19); Model amphipathy having no sequence homology with ApoA-I called LAP peptide consisting of 16-24 amino acid residues Peptides (for example, see Non-patent Document 20); peptides consisting of 18-24 amino acid residues having no sequence homology with the ApoA-I helix (for example, see Non-patent Document 21); human ApoA-I helix sequences Peptides having 22 amino acid residues in common based on the above (see, for example, Non-Patent Documents 22 and 23). However, none of the ApoA-I pseudopeptides prepared so far show the same level of activity as ApoA-I, and none show the activity that can be said to be useful as a pharmaceutical (for example, Patent Document 1). reference).

In addition to the ApoA-I pseudopeptide described above, a number of ApoA consisting of a “core” peptide consisting of 15 to 29 amino acid residues, preferably 22 amino acid residues, that form an amphipathic α-helix in the presence of lipids. -I agonists have been produced (see, for example, Patent Documents 1 and 5). These ApoA-I agonists are 22-mer consensus sequences (Pro (P) にとって Val (V) Leu (L) Asp (D) Glu (E) Phe (F) proposed to be critical for activity. Arg (R) Glu (E) Lys (K) Leu (L) Asn (N) Glu (E) Glu (E) Leu (L) Glu (E) Ala (A) Leu (L) Lys (K) Gln ( Q) Lys (K) Leu (L) Lys (K): SEQ ID NO: 2) (cited in the cited literature as SEQ ID NO: 75, hereinafter referred to as “common 22-mer”). It is described that synthetic peptides showing activity close to or exceeding the activity of natural ApoA-I were obtained by changing amino acid residues (see, for example, Patent Documents 1 and 8). Furthermore, by replacing three charged amino acid residues (Glu-5, Lys-9 and Glu-13) in this common 22-mer peptide with hydrophobic leucine residues, ApoA- It is described that a pseudo peptide having the structure and functional characteristics of I was obtained (see, for example, Patent Documents 1 and 8). It must be said that these pseudo-peptides still have no activity that is useful as pharmaceuticals.

In addition, the apolipoprotein ApoA-I (ApoA-I), although only its central helix can promote lipid excretion mediated by ABCA1 (ATP-binding castle transporter A1) In order to exert a functional interaction, a sufficiently long amino acid sequence is required between ApoA-I and ABCA1, and it has been reported that the length is 220-231 residues. (Non-patent Document 24).

US Patent No. 6004925 US Patent No. 5089602 French patent 2343251 WO / 1994/013819 WO / 1999/016409 US Patent No. 7053049 US Pat. Nos. 5,128,318 and 5,652,339 Special table 2001-517710, Special table 2001-518282, Special table 2003-525565, Special table 2003-525840, Special table 2001-522781

Therefore, in view of the prior art, the present inventors have studied a synthetic peptide having an activity similar to that of human apolipoprotein AI (ApoA-I). As a result, the amino acids constituting the central helix of ApoA-I The present invention was completed by finding that certain synthetic peptides in which a part of the amino acid residues of the sequence were mutated had the ability to export cholesterol. Furthermore, the present inventors have found that such a synthetic peptide has not only the ability to export cholesterol but also the ability to produce HDL, thereby completing the present invention.

Therefore, the present invention relates to a cholesterol export peptide having a cholesterol export ability and a HDL production ability, in which a part of the amino acid residues of the amino acid sequence constituting the central helix of ApoA-I is mutated. The purpose is to provide.

More specifically, an object of the present invention is to provide a cholesterol export peptide represented by the following general formula [I].

Moreover, an object of the present invention is to provide a pharmaceutical composition containing the above cholesterol export peptide [I] as an active ingredient.

This invention provides a cholesterol export peptide, which is a certain synthetic peptide having a cholesterol export ability, in which a part of the amino acid residues of the amino acid sequence constituting the central helix of ApoA-I is mutated.

In addition, as a preferred embodiment, the present invention provides a cholesterol export peptide which is a synthetic peptide having a cholesterol export capability and an HDL production capability.

Furthermore, the present invention has a preferred embodiment represented by the general formula [I]:

H-X1-X2-X3-His-Leu-X4-Thr-Leu-X5-Glu-Lys-Ala-
X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-
X18-OH [I]

[Where X1 is Ala, single bond (“-”) or general formula [Ia]:

X1e-X1d-X1c-X1b-X1a- [Ia]

(In the formula, X1a means Ala or a single bond ("-"), X1b means Lys, Arg or a single bond ("-"), and X1c means Ala or a single bond ("-"). , X1d means His or a single bond ("-"), and X1e means Tyr or a single bond ("-").)
X2 means Thr, Leu, Lys or Ser,
X3 means Glu, Thr or Asp
X4 means Ser, Phe or Lys
X5 means Ser, Tyr, Trp, Phe or Gly
X6 represents a single bond ("-"), Lys, Leu or Arg,
X7 represents a single bond ("-"), Pro or Lys,
X8 means single bond ("-") or Ala,
X9 means a single bond ("-"), Phe or Leu,
X10 means a single bond ("-"), Glu, Gln or Asp,
X11 means single bond ("-") or Asp,
X12 means single bond ("-") or Leu,
X13 represents a single bond ("-"), Arg, Leu or Gly,
X14 means single bond ("-"), Gln or His,
X15 means a single bond ("-"), Gly, Lys or Ser,
X16 means single bond ("-"), Leu or His,
X17 is a single bond ("-"), Leu, Met or general formula [Ib]:

X17a-X17b-X17c-X1d [Ib]
(In the formula, X17a means a single bond ("-"), Leu or Met, X17b means Pro, Tyr or a single bond ("-"), X17c means Val or a single bond ("-") As well as X1d means Leu or a single bond ("-"))

However, when the symbols X6-X17, X1a-X1e and the symbols X17a-X17d all mean single bonds ("-"), these single bonds as a whole mean one single bond ("-"). At the same time, X1 is Ala, X2 is Thr, X3 is Glu, X4 is Ser, X5 is Ser, X6 is Lys, X7 is Pro, X8 is Ala, X9 is Leu, X10 is Glu, X11 is Asp, X12 Is Leu, X13 is Arg, X14 is Gln, X15 is Gly, X16 is Leu, and X17 is Leu. ]
Or a peptide represented by the general formula [II]:

A -X20 (X21-OH)-A [II]
[Wherein A represents the general formula [IIa]:
H-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-
X33-X34-X35-X36-X37-X38-X39-X40-X41-X42-X43-X44-
X45-X46- [IIa]

(Wherein X22 means Ala or Val, X23 means Thr, Leu, Lys or Ser, X24 means Glu, Thr or Asp, X25 means His or Ser, and X26 means Leu or Means Phe, X27 means Ser, Phe or Lys, X28 means Thr or Val, X29 means Leu or Ser, X30 means Ser, Gly, Phe, Tyr or Trp, X31 Means Glu or Leu, X32 means Lys or Ser, X33 means Ala, X34 means Lys, Leu, Arg or single bond (“-“), X35 means Pro, Glu, Lys Or a single bond ("-"), X36 means Ala, Glu or a single bond ("-"), X37 means Leu, Tyr or a single bond ("-"), X38 means Glu, Gln, Asp, Thr or single bond ("-"), X39 means Asp, Lys or single bond ("-"), X40 means Leu, Lys or single bond ("-") , X41 means Arg, Gly, Leu or single bond ("-"), X42 means Gln, Leu, Lys, His or bond ( "-"), X43 means Gly, Leu, Lys, Ser or bond ("-"), X44 means Leu, His or bond ("-")),
X20 means an amino acid residue such as Lys,
X21 means an amino acid residue such as Leu or Ala. )
Or a general formula [III]:

(A) ₂ -X22 (X23-OH)-(A) ₂ [III]
Wherein A has the same meaning as above,
X22 means an amino acid residue such as Lys, and
X23 means an amino acid residue such as Leu or Ala. )
A cholesterol export peptide comprising a tetramer represented by the formula:

Moreover, the present invention provides a pharmaceutical composition containing the above cholesterol export peptide as an active ingredient.

The cholesterol-carrying peptide of the present invention is effective in preventing and treating cardiovascular diseases because it has both the effect of carrying out cholesterol and the ability to produce HDL.

Diagram showing the ability of peptide (FAMP) and ApoA-I to pull cholesterol and the ability of peptide (FAMP) to pull cholesterol significantly upon stimulation with TO901317 and 9cis-retinoic acid (9cisRA) in human A172 cells (Example 3). FIG. 10 is a graph showing the cholesterol pulling action of a peptide (FAMP-5: Type 2S9Y) in a Chinese hamster ovary-derived cell (CHO cell) ldlA7 strain (Example 4). The figure which shows the cholesterol pulling-out ability by the healthy subject origin cell and human ABCA1 deficiency patient origin cell in the human peripheral blood monocyte origin macrophage using the peptide (FAMP) synthesize | combined in Example 1. FIG. FIG. 11 shows the ability of human A172 cells to extract cholesterol in the absence of or with stimulation of TO901317 and 9cis-retinoic acid for each peptide (20 μg / ml) (Example 6). The figure which shows the cholesterol extraction ability under stimulation of TO901317 and 9cis-retinoic acid of each peptide (20 μg / ml) by human A172 cells (Example 7). The figure which shows the analysis result of the human plasma lipoprotein by the capillary electrophoresis method in the presence and absence of the peptide (FAMP) (2 mg / ml) prepared in Example 1. Example 9 The figure which shows the analysis result of the plasma lipoprotein by the high performance liquid chromatography (HPLC) when the peptide is continuously administered to the mouse for 10 days (Example 10). The figure which shows the analysis result of mouse | mouth plasma lipoprotein by the capillary-electrophoresis method at the time of administering a peptide intravenously to a mouse | mouth. (Example 11) The figure which shows the analysis result of the mouse | mouth plasma lipoprotein by the capillary electrophoresis method when a peptide is intraperitoneally administered to a mouse | mouth (Example 11). FIG. 10 shows the ability of each peptide (20 μg / ml) to be extracted by human A172 cells under stimulation with TO901317, LXR agonist, 9-cis-retinoic acid, and RXR agonist (Example 12). Peptide (FAMP-5) に対する on human A172 cells and apoA-I TO901317, LXR agonist (5μM), 9-cis-retinoic acid, in the absence or presence of Probunol (10 μM) upon stimulation with RXR agonist The figure which shows the cholesterol extraction ability (Example 13). The figure which shows the cholesterol extraction ability under the stimulation by TO901317, LXR agonist (5μM), 9-cis-retinoic acid, RXR agonist of each peptide for human A172 cells (Example 14). Diagram showing the ability of each peptide to pull cholesterol in the absence or presence of Probunol® (10 μM) by stimulation with TO901317, LXR®agonist® (5 μM), 9-cis-retinoic acid, RXR®agonist on human A172 cells Example 14). The figure which shows the experimental result using the agarose riboprotein electrophoresis of a peptide (FAMP-5) (Example 15). The figure (Example 16) which shows the blood flow increase with respect to a mouse | mouth leg ischemia model of a peptide (FAMP-5) sputum. The figure which shows that the fecal excretion of 3H-cholesterol in a macrophage increased significantly (Example 17).

The cholesterol export peptide according to the present invention has the general formula [I]:
H-X1-X2-X3-His-Leu-X4-Thr-Leu-X5-Glu-Lys-Ala-
X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-
X18-OH [I]

[Wherein X1 is Ala, single bond ("-") or general formula [Ia]:
X1e-X1d-X1c-X1b-X1a- [Ia]

However, when the symbols X1a-X1e and X17a-X17d both mean a single bond ("-"), these single bonds as a whole mean one single bond ("-"), and At the same time, X1 is Ala, X2 is Thr, X3 is Glu, X4 is Ser, X5 is Ser, X6 is Lys, X7 is Pro, X8 is Ala, X9 is Leu, X10 is Glu, X11 is Asp, X12 is Leu, X13 Except Arg, X14 is Gln, X15 is Gly, X16 is Leu, and X17 is Leu. ]
Or a peptide represented by the general formula [II]:

A -X20 (X21-OH)-A [II]
[Wherein A represents the general formula [IIa]:
H-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-
X33-X34-X35-X36-X37-X38-X39-X40-X41-X42-X43-
X44-X45-X46- [IIa]

(Wherein X22 means Ala or Val, X23 means Thr, Leu, Lys or Ser, X24 means Glu, Thr or Asp, X25 means His or Ser, and X26 means Leu or Means Phe, X27 means Ser, Phe or Lys, X28 means Thr or Val, X29 means Leu or Ser, X30 means Ser, Gly, Phe, Tyr or Trp, X31 Means Glu or Leu, X32 means Lys or Ser, X33 means Ala, X34 means Lys, Leu, Arg or single bond (“-“), X35 means Pro, Glu, Lys Or a single bond ("-"), X36 means Ala, Glu or a single bond ("-"), X37 means Leu, Tyr or a single bond ("-"), X38 means Glu, Gln, Asp, Thr or single bond ("-"), X39 means Asp, Lys or single bond ("-"), X40 means Leu, Lys or single bond ("-") , X41 means Arg, Gly, Leu or single bond ("-"), X42 means Gln, Leu, Lys, His or bond ( "-"), X43 means Gly, Leu, Lys, Ser or bond ("-"), X44 means Leu, His or bond ("-")),
X20 means an amino acid residue such as Lys, and
X21 means an amino acid residue such as Leu or Ala. )
Or a general formula [III]:

(A) ₂ -X22 (X23-OH)-(A) ₂ [III]
Wherein A has the same meaning as above,
X22 means an amino acid residue such as Lys, and
X23 means an amino acid residue such as Leu or Ala. )
It is a tetramer represented by

As used herein, the term “amino acid residue” refers to an amino acid whose N-terminal amino group (—NH ₃ ) is an imino group (—NH ₂ —) and whose C-terminal carboxyl group (—COOH) is a carbonyl group (—CO It means a divalent amino acid residue in the state of-). An amino acid residue has a peptide bond (—CO—NH) with its N-terminal imino group (—NH ₂ —) bonded to the C-terminal carbonyl group (—CO—) of another amino acid residue adjacent to the N-terminal side. -), And the C-terminal carbonyl group (—CO—) binds to the N-terminal imino group (—NH ₂ —) of another amino acid residue adjacent to the C-terminal side to form a peptide bond (— CO—NH—) are formed, each linked to a different amino acid residue. Therefore, the symbol (“-”) between each amino acid residue represented by the symbol X and its adjacent amino acid residue means a peptide bond connector. If there is no adjacent amino acid residue, the N-terminal imino group (—NH ₂ —) of the amino acid residue is bonded to a hydrogen atom (H—) to form an amino group, while the C-terminal carbonyl group is (—CO—) binds to a C-terminal hydroxy group (—OH) to form a carboxyl group (—CO—OH). In other words, if there is no amino acid residue in the symbol X1b adjacent to the amino acid residue represented by the symbol X1a, the symbol X1b and the subsequent symbols X1c-X1d are all single bonds, and the amino acid residue represented by X1a The N-terminal imino group (—NH ₂ —) of N is bonded to a hydrogen atom (H—) to form an amino group. On the other hand, similarly, when there is no amino acid residue in the symbol X17b adjacent to the amino acid residue represented by X17a, the symbol X17b and the subsequent symbols X17c-X17d all become single bonds, and the amino acid residue represented by X17a The C-terminal carbonyl group (—CO—) of the group is bonded to the C-terminal hydroxy group (—OH) to form a carboxyl group (—CO—OH).

In the above general formula, when each of X6-X17, X1a-X1e and X17a-X17d means a single bond ("-"), it constitutes one continuous single bond ("-") as a whole. That means.

Among the cholesterol export peptides according to the present invention, preferable peptides include, for example, vetide having the following amino acid sequence.

(Bebutide No. 1) 11 amino acid residues (human type) (SEQ ID No. 2)
H-Thr-Glu-His-Leu-Ser-Thr-Leu-Ser-Glu-Lys-Ala-OH
(Bebutide No. 2) 12 amino acid residues (human type) (SEQ ID NO: 3)
H-Ala-Thr-Glu-His-Leu-Ser-Thr-Leu-Ser-Glu-Lys-Ala-OH
(Bebutid No. 3) 23 amino acid residues (human type) (SEQ ID No. 4)
H-Tyr-His-Ala-Lys-Ala-Thr-Glu-His-Leu-Ser-Thr-Leu-Ser-Glu-Lys-Ala-Lys-Pro-Ala-Leu-Glu-Asp-Leu-OH
(Bevetide No. 4) Type1 (SEQ ID No. 5)
H-Ala-Leu-Glu-His-Leu-Phe-Thr-Leu-Ser-Glu-Lys-Ala-Lys-Lys-Ala-Leu-Glu-Asp-Leu-Leu-Leu-Lys-Leu-Leu- OH
(Bevetide number 5) Type2 (FAMP) (SEQ ID NO: 6)
H-Ala-Leu-Glu-His-Leu-Phe-Thr-Leu-Ser-Glu-Lys-Ala-Leu-Lys-Ala-Leu-Glu-Asp-Leu-Leu-Lys-Lys-Leu-Leu- OH
(Bevetide No. 6) FAMP-5 (Type2S9Y) (SEQ ID NO: 7)
H-Ala-Leu-Glu-His-Leu-Phe-Thr-Leu-Tyr-Glu-Lys-Ala-Leu-Lys-Ala-Leu-Glu-Asp-Leu-Leu-Lys-Lys-Leu-Leu- OH
(Bevetide number 7) Type2S9W (SEQ ID NO: 8)
H-Ala-Leu-Glu-His-Leu-Phe-Thr-Leu-Trp-Glu-Lys-Ala-Leu-Lys-Ala-Leu-Glu-Asp-Leu-Leu-Lys-Lys-Leu-Leu- OH
(Bevetide number 8) Type3 (SEQ ID NO: 9)
H-Ala-Thr-Glu-His-Leu-Ser-Thr-Leu-Ser-Glu-Lys-Ala-Leu-Lys-Ala-Phe-Glu-Asp-Leu-Leu-Lys-Lys-Leu-Leu- OH
(Bevetide number 9) Type4 (SEQ ID NO: 10)
H-Ala-Thr-Glu-His-Leu-Ser-Thr-Leu-Ser-Glu-Lys-Ala-Lys-Pro-Ala-Leu-Glu-Asp-Leu-Leu-Lys-Lys-Leu-Leu- OH
(Bebutide No. 10) 25 amino acid residues (Type2NY) (SEQ ID NO: 11)
H-Ala-Leu-Glu-His-Leu-Phe-Thr-Leu-Ser-Glu-Lys-Ala-Leu-Lys-Ala-Leu-Glu-Asp-Leu-Leu-Lys-Lys-Leu-Leu- Tyr-OH
(Bebutide No. 11) 27 amino acid residues (Rattus type) type-J5 (SEQ ID No. 12)
H-Ala-Ser-Asp-His-Leu-Lys-Thr-Leu-Gly-Glu-Lys-Ala-Lys-Pro-Ala-Leu-Asp-Asp-Leu-Gly-Gln-Gly-His-Met- Pro-Val-Leu-OH
(Bebutide No. 12) 24 amino acid residues (Murine type) type-J6 (SEQ ID NO: 13)
H-Arg-Ala-Lys-Thr-His-Leu-Lys-Thr-Leu-Gly-Glu-Lys-Ala-Arg-Pro-Ala-Leu-Gln-Asp-Leu-Arg-His-Ser-Leu- OH
(Bebutide No. 13) 24 amino acid residues (human type) (Original: SEQ ID NO: 14)
H-Ala-Thr-Glu-His-Leu-Ser-Thr-Leu-Ser-Glu-Lys-Ala-Lys-Pro-Ala-Leu-Glu-Asp-Leu-Arg-Gln-Gly-Leu-Leu- OH

(Bebutide No. 14) Dimer (FAMP-5-Duo) (SEQ ID NO: 15)

(Bebutide No. 15) Dimer (Human type original (221-240)-Duo) (SEQ ID NO: 16)

(Bebutide No. 16) Tetramer (FAMP-5-Quad)

The cholesterol export peptide according to the present invention can be prepared according to a synthetic chemical technique conventionally used in the art. Synthetic chemical methods include, for example, carbodiimide condensing agents such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) as condensing agents, (1H -Benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate (BOP), 1- [bis (dimethylamino) methylene] -1H-benzotriazolium-3-oxide hexafluorophosphate -Condensation method using condensing agent such as HBTU, active ester method using ester with nitrophenol, N-hydroxysuccinimide, pentafluorophenol, etc., chloroformate, carboxylic acid chloride, etc. Prepared using a liquid phase method such as the mixed acid anhydride method, a solid phase peptide synthesis method such as the Fmoc method and the Boc method. Can.

The cholesterol export peptide according to the present invention increases the high density lipoprotein (HDL) concentration, activates the cholesterol acyltransferase (LCAT), and further activates the reverse cholesterol transport system (RCT). It is useful to promote and enhance the efflux of cholesterol. Therefore, the cholesterol export peptide of the present invention is effective in the prevention and treatment of cardiovascular diseases such as atherosclerosis and hyperlipidemia such as hypercholesterolemia and cardiovascular disease related disorders. It is.

In order to prevent or treat such cardiovascular diseases and disorders, the cholesterol export peptide of the present invention may be used alone or in combination with one or more thereof, or for the prevention or treatment of cardiovascular diseases and disorders. It can be used as a pharmaceutical composition in combination with other drugs that can be used, for example, cholesterol-lowering drugs such as statins and niacin and fibrates.

The pharmaceutical composition of the present invention can also be formulated as a peptide-lipid complex. When formulated as a peptide-lipid complex, usable lipids may be, for example, saturated or unsaturated lipids, and may be natural or synthetic lipids. Examples of such lipids include egg phosphatidylcholine, soybean phosphatidylcholine, dilaurylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, 1-palmitoyl-2-oleylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoylcholine. Phosphatidylcholines such as -2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine; phosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleophosphatidylethanol, etc. The ho Phosphatidylglycerols such as phosphatidylglycerol, diphosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol Phosphatidic acids such as phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid; phosphatidyl serines such as phosphatidyl serine, dimyristoyl phosphatidyl serine, dipalmitoyl phosphatidyl serine, brain phosphatidyl serine; sphingomyelin, brain sphingomyelin, dipalmitoyl Sphingomyelin such as sphingomyelin, distearoyl sphingomyelin Phosphatidylinositol; sphingolipids such as sphingosine 1-phosphate (SIP); galactocerebroside, ganglioside, cerebroside, (1,3) -D-mannosyl- (1,3) diglyceride, amino Examples include phenylglycoside, 3-cholesteryl-6 ′-(glycosylthio) hexyl ether glycolipid, and cholesterol, and derivatives thereof.

The pharmaceutical composition according to the present invention may further contain a pharmacologically acceptable carrier in addition to the cholesterol export peptide of the present invention and other drugs. As such a pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as a raw material for preparations can be used. Examples of such carrier substances include excipients, lubricants, binders, and disintegrants in solid preparations, and solvents, dispersants, preservatives, isotonic agents, and dissolution aids in liquid preparations. , Suspending agents, stabilizers, buffers, soothing agents and the like. Moreover, in the pharmaceutical composition of this invention, formulation additives, such as antiseptic | preservative, antioxidant, a coloring agent, a sweetener, can also be used, for example.

In the solid preparation of the pharmaceutical composition of the present invention, examples of excipients include lactose, sucrose, starch, pregelatinized starch, D-mannitol, D-sorbitol, dextrin, crystalline cellulose, carboxymethyl Cellulose sodium, gum arabic, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, magnesium metasilicate aluminate and the like. Examples of the lubricant include talc, magnesium stearate, calcium stearate, colloidal silica, polyethylene glycol 6000, and the like. Examples of binders include gelatin, pregelatinized starch, sucrose, gum arabic, crystalline cellulose, methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose sodium, sucrose, D -Mannitol, trehalose, dextrin, pullulan, polyvinylpyrrolidone and the like. Examples of the disintegrant include lactose, sucrose, starch, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, sodium carboxymethyl starch, and light anhydrous silicic acid.

In the liquid preparation of the pharmaceutical composition of the present invention, examples of the solvent include an aqueous solvent such as distilled water, physiological saline, Ringer's solution, a tree solvent such as alcohol, propylene glycol, polyethylene glycol, or the like. And oily solvents such as sesame oil, corn oil, olive oil, and vegetable oils such as cottonseed oil. Examples of the dispersant include polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate and the like. Examples of preservatives include methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, phenol and the like. Examples of tonicity agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like. Examples of solubilizers include ethanol, polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, triethanolamine, trisaminomethane, cholesterol, sodium carbonate Sodium citrate, sodium salicylate, sodium acetate and the like. Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, polyvinyl alcohol, polyvinyl Hydrophilic polymer compounds such as pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polysorbates, polyoxyethylene hydrogenated castor oil, etc. Is mentioned. Examples of stabilizers include human serum albumin. Examples of the buffer include buffer solutions such as phosphate, acetate, carbonate, citrate and the like. Examples of soothing agents include benzyl alcohol.

Examples of preservatives include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfite and ascorbate. Examples of colorants include natural pigments such as β-carotene, chlorophyll, bengara, water-soluble food tar pigments such as

food red

2, 3,

food yellow

4, 5,

food blue

1, 2, etc. Examples thereof include water-insoluble lake dyes such as aluminum salts of water-soluble food tar dyes. Examples of sweetening agents include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.

The pharmaceutical composition according to the present invention can be administered by oral route or parenteral route. In order to administer the pharmaceutical composition of this invention by the oral route of administration, for example, liquid dosage forms such as solutions, emulsions, syrups, suspensions, capsules such as tablets, soft capsules, microcapsules, granules It can be administered in a solid dosage form such as an agent, powder, powder, or troche. On the other hand, in order to administer the pharmaceutical composition of the present invention via a parenteral route, for example, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, infusions such as drops, transdermal preparations, etc. It may be administered in a dosage form such as an external preparation such as an ointment, a suppository, a pellet, a nasal preparation, an inhalant, or an eye drop. These preparations may be controlled-release preparations such as immediate-release preparations or sustained-release preparations (for example, sustained-release microcapsules).

The pharmaceutical composition of the present invention can be produced by a method commonly used in the technical field relating to the preparation, for example, a method described in the Japanese Pharmacopoeia. For example, an oral preparation is compression-molded by adding an excipient, a disintegrant, a binder or a lubricant to an active ingredient, and then, if necessary, for the purpose of taste masking, entericity or sustainability. It can be produced by coating using a coating base by a method known per se. Examples of the coating base include sugar coating base, water-soluble film coating base, enteric film coating base, sustained-release film coating base and the like.

Among such coating bases, as sugar coating base, for example, sucrose, talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like can be used. Examples of the water-soluble film coating base include cellulose-based polymers such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, methylhydroxyethyl cellulose; -Synthetic polymers such as rudiethylaminoacetate and polyvinylpyrrolidone; polysaccharides such as pullulan can be used. Examples of enteric film coating bases include hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, carboxymethyl ethyl cellulose, and cellulose acetate phthalate. Cellulose polymers; acrylic acid polymers such as methacrylic acid copolymers; natural products such as shellac can be used. As the sustained-release film coating base, for example, a cellulose polymer such as ethyl cellulose; and an acrylic acid polymer such as aminoalkyl methacrylate copolymer can be used.

In addition, the injection can be produced by a method known per se by dissolving, suspending or emulsifying the active ingredient in an aqueous solvent together with a dispersant, a preservative, an isotonic agent and the like. In the production of such an injection, additives such as a solubilizing agent, a stabilizer and a soothing agent may be used as desired.

Since the pharmaceutical composition according to the present invention contains the cholesterol export peptide of the present invention as the main active ingredient, it is possible to carry out cholesterol export, increase serum HDL concentration and LCAT activation, and promote RCT. It is useful and can be used for the prevention and treatment of cardiovascular diseases and related disorders, particularly in mammals including humans. More specifically, the pharmaceutical composition of the present invention can be used for, for example, cardiac diseases such as angina pectoris, hypertension, hyperlipidemia such as hypercholesterolemia, and arteriosclerosis such as atherosclerosis. Because of its vascular disease and angiogenic action, it is also effective for obstructive arteriosclerosis, Buerger's disease of lower limb ischemia, Alzheimer's disease with similar risk, and the like. Furthermore, it is effective for cardiovascular disease-related disorders such as restenosis due to atherosclerotic plaque that develops as a result of medical treatment such as balloons and stents.

The single dose of the cholesterol export peptide and pharmaceutical composition according to the present invention varies depending on the patient's medical condition, physical condition, method of administration, etc., but in parenteral administration, it is about 0 as an active ingredient per kg adult body weight. .1 to 100 mg, preferably about 0.5 to 50 mg, more preferably about 1 to 25 mg, and the daily dose is preferably 1 to 3 times by intravenous or intramuscular injection. For oral or nasal administration, the single dose is about 1 to 100 mg, preferably about 2 to 50 mg as an active ingredient per kg body weight of an adult, and the daily dose is divided into 1 to 3 doses. It is good to do.

The present invention will be described in more detail with reference to the following examples. However, the following examples are not described in any way with the intention of limiting or limiting the present invention, and the present invention is merely illustrated and described in more detail. It should be understood that it is only to do. In addition, it should be understood that the scope of the present invention includes variations and improvements of the following examples.

Peptide No. 5 (FAMP: SEQ ID No. 6) Synthesis Method Peptides were synthesized at 0.25 mmol scal using an Applied Biosystems model 433A peptide automatic solid phase synthesizer. Amino acids are Fmoc-amino acid, Fmoc-X-PEG-Alko-Resin is used as resin, 20% Piperidine / N-Methyl-2-pyrrolidone is used as a de-Fmoc reagent, and HBTU / HOBt in DMF is used as an active condensing agent. The coupling was synthesized repeatedly. The sample was transferred to a manual solid phase synthesis vessel, washed several times with DCM, and then dried in a desiccator under reduced pressure overnight. After drying, add the cleaving reagent (0.25 ml EDT / 0.25 H ₂ O / TFA 9.5 ml) and stir for 2 hours to cleave the peptide from the resin and finally deprotect the side chain. The solution was filtered, and the filtrate was transferred to an eggplant flask and concentrated with nitrogen gas. Crystallization was performed using cold ether, and after 10 times decantation, this was collected by filtration to obtain a crude peptide. 50 mg of the obtained crude peptide was dissolved in 3 ml of secondary water, and this aqueous solution was fractionated by gel chromatography using Sephadex G-25. The absorbance (260 nm) of the fractionated fraction was measured, and each peak portion was collected and lyophilized to obtain the target peptide. Identification was performed by MALDI TOF-MS.

Peptide number (1: SEQ ID NO: 2), peptide number (2: SEQ ID NO: 3), peptide number (3: SEQ ID NO: 4), peptide number (4: SEQ ID NO: 5), peptide number (6: SEQ ID NO: 7), Peptide number (7: SEQ ID NO: 8), peptide number (8: SEQ ID NO: 9), peptide number (9: SEQ ID NO: 10), peptide number (10: SEQ ID NO: 11), peptide number (11: SEQ ID NO: 12), Peptide number (12: Sequence number 13), Peptide number (13: original: Sequence number 14), Peptide number (14: FAMP-5-Duo: Sequence number 15), Peptide number (15: Human type original (221- 240) Each peptide represented by-Duo: SEQ ID NO: 16) and peptide number (16: FAMP-5-Quad) was synthesized and confirmed in substantially the same manner as in Example 1.

Using the peptide (FAMP) synthesized in Example 1, the cholesterol withdrawal action in human A172 cells was examined.

In this example, human A172 cells were contacted with peptide (FAMP) (20 μg / ml) and human serum-derived lipid-removed ApoA-I (20 μg / ml) as a comparison, and left for 4 hours. After 4 hours, the amount of cholesterol exuded from human A172 cells was measured to examine the ability of peptide (FAMP) and ApoA-I to extract cholesterol (left side of FIG. 1). As a control, only 0.2% bovine serum albumin (BSA) was used. As a result, peptide (FAMP) and ApoA-I have the ability to extract cholesterol, and the ability of peptide (FAMP) to extract cholesterol is significantly superior to that of ApoA-I. It was.

In addition, it was found that this cholesterol withdrawal action is markedly increased by stimulation with TO901317 and 9cis-retinoic acid (9cisRA) (right side of FIG. 1). As a result, it has been clarified that peptide (FAMP) alone has a cholesterol-removing action and also has an HDL-forming action.

FIG. 1 shows the results of examining the cholesterol withdrawal ability of each peptide produced in Example 2 in substantially the same manner as in Example 3. Saline was used as a control.

Using the peptide (FAMP) synthesized in Example 1, the action of pulling out cholesterol in the ldlA7 strain of Chinese hamster ovary-derived cells (CHO cells) was examined.

In this example, in the CHO cell ldlA7 strain, plasmids in which human ABCA1 cDNA was inserted into pcDNA3.1 vector (mock) and pcDNA3.1 vector were respectively transfected using the lipofection method. Mock and ABCA1 cDNA cDNA-introduced CHO cells were left in contact with peptide (FAMP-5) (20 μg / ml) and human serum-derived lipid-removed ApoA-I (20 μg / ml) as a comparison for 4 hours. After 4 hours, the amount of cholesterol exuded from the cells was measured to examine the ability of the peptide (FAMP) and ApoA-I to extract cholesterol (FIG. 2). As a control, only 0.2% BSA was used. As a result, both Mock and ABCA1 cDNA gene-introduced CHO cells showed a significant cholesterol-removing action for both peptide (FAMP) and human serum ApoA-I, but in ABCA1 cDNA gene-introduced CHO cells, peptide (FAMP) Increased the cholesterol withdrawal effect more significantly. This reveals that peptide (FAMP) has a cholesterol-removing action as a peptide alone and also has a HDL-forming action depending on ABCA1 transporter.

Using the peptide synthesized in Example 1 (FAMP), the cholesterol withdrawal action in human peripheral blood monocyte-derived macrophages was examined in substantially the same manner as in Example 4.

In this example, in a macrophage derived from human peripheral blood monocytes, cells derived from healthy subjects and cells derived from patients with ABCA1 deficiency were compared with peptide (FAMP) (20 μg / ml) and human serum-derived lipid-removed ApoA-I ( 20 μg / ml) and left for 4 hours. After 4 hours, the amount of cholesterol exuded from the cells was measured to examine the ability of the peptide (FAMP) and ApoA-I to extract cholesterol (FIG. 3). As a control, only 0.2% BSA was used. As a result, in cells derived from healthy subjects, both peptide (FAMP) and human serum ApoA-I showed significant cholesterol withdrawal, but in cells derived from ABCA1 deficiency, peptide (FAMP) was found to be cholesterol. Although the withdrawal action was retained, the cholesterol withdrawal action of human serum ApoA-I was deficient. This indicates that the peptide (FAMP) has a cholesterol-removing action with the peptide alone and also has an HDL-forming action independent of the ABCA1 transporter.

In the same manner as in Example 3, the ability of pulling out cholesterol for each of peptide number 5 (Type 2), peptide number 6 (FAMP-5) and peptide number 9 (Type 4) was measured using human A172 cells.

That is, each peptide (20 μg / ml) was brought into contact with human A172 cells and incubated for 4 hours, and the amount of cholesterol exuded from human A172 cells was measured to examine the ability to extract cholesterol. As a control, only 0.2% bovine serum albumin (BSA) was used, and ApoA-I and peptide number 13 (original) were used for comparison. The results are shown on the left side of FIG.

On the other hand, each peptide (20μg / ml) was contacted with TO901317 and 9cis-retinoic acid in human A172 cells and incubated for 4 hours, and then the amount of cholesterol leached from human A172 cells was measured. Then I investigated the ability to pull out cholesterol. The results are shown on the right side of FIG.

From these results, it was revealed that Peptide No. 6 (FAMP-5) has a significantly superior cholesterol withdrawal ability.

In the same manner as in Example 1, each peptide (20 μg / ml) was contacted with human A172 cells under stimulation with TO901317 and 9cis-retinoic acid and incubated for 4 hours, and then the cholesterol exuded from human A172 cells. The amount was measured to determine the cholesterol withdrawal ability. The results are shown in FIG.

In this example, human plasma lipoprotein was analyzed by capillary electrophoresis.

In vitro, human plasma was incubated at 17 ° C. for 150 minutes in the presence and absence of the peptide (FAMP) prepared in Example 1 (2 mg / ml). The incubated human plasma was analyzed by capillary electrophoresis (FIG. 6). From this analysis result, it was recognized that the preβ-HDL subfraction was significantly increased in human plasma incubated in the presence of peptide (FAMP). This result shows that the peptide of this invention has the effect | action which born HDL. Capillary-electrophoresis was performed according to the technique of Zhangbo et al. (Zhang, B. et al.).

In this example, plasma lipoprotein was analyzed by high performance liquid chromatography (HPLC) when the peptide was continuously administered to mice for 10 days. Changes in blood lipid profile after continuous intraperitoneal administration of peptide (FAMP) (2 mg / ml) and physiological saline for 10 days to C57BL6 mice were measured by high performance liquid chromatography (HPLC). As a result, it was recognized that the HDL subfraction was markedly increased by continuous administration of the peptide (FAMP) intraperitoneally for 10 days (FIG. 7). This analysis result shows that the peptide of the present invention has an action of generating HDL even in vivo.

In this example, mouse plasma lipoprotein was analyzed by capillary electrophoresis when the peptide was acutely administered to mice. After the peptide (FAMP) (2 mg / ml) was intravenously and intraperitoneally administered to C57BL6 mice, changes in blood lipid profile after 30 minutes and 18 hours were measured by capillary electrophoresis (FIG. 8 and FIG. 8). FIG. 9). As a result, it was confirmed that the peptide (FAMP) markedly increased the HDL subfraction even in intravenous administration and intraperitoneal administration. This analysis result shows that the peptide of the present invention has an action of generating HDL even in vivo. Capillary electrophoresis was performed in the same manner as in Example 9.

In the same manner as in Example 1, each peptide (20 μg / ml) was stimulated with TO901317, LXR agonist (5μM), 9-cis-retinoic acid, RXR agonist (5μM) in human A172 cells and incubated for 4 hours. Then, the amount of cholesterol exuded from human A172 cells was measured to examine the ability to extract cholesterol. Saline was used as a control. The results are shown in FIG.

In the same manner as in Example 12, the peptide FAMP-5 (type2S9Y) or apoA-I (20µg / ml) was added to TO901317, LXR agonist (5µM), 9-cis-retinoic acid, RXR agonist (5µM) in the same manner as in Example 12. After stimulation, the amount of cholesterol exuded from human A172 cells was measured by incubating in the absence or presence of Probunol® (10 μM) for 4 hours to examine the ability to extract cholesterol. Saline was used as a control. The results are shown in FIG.

In the same manner as in Example 12, each peptide (FAMP-5, FAMP-5-Duo, Human type original (221-240, Human type original (221-240) -Duo) (20 μg / ml) was added to human A172 cells. After stimulation with TO901317, LXR agonist (5μM), 9-cis-retinoic acid, RXR agonist (5μM), incubate for 4 hours in the absence or presence of Probunol (10μM) and leach from human A172 cells The amount of cholesterol was measured to examine the ability to extract cholesterol, and physiological saline was used as a control, and the results are shown in FIGS.

Experiments using agarose riboprotein electrophoresis were performed. Peptide (FAMP-5) (0.2 mg / ml ～ 2 mg / ml) was incubated with human plasma at 37 ° C for 150 minutes and then subjected to agarose riboprotein electrophoresis. Prominent production of rich pre-β was observed. The results are shown in FIG.

An experiment using a mouse lower limb ischemia model was conducted. After ligating the left femoral artery of the mouse, PBS or peptide (FAMP-5) (10 mg / ml or 50 mg / ml was intramuscularly injected into the affected (left) limb on the first, third, and fifth days. Lower limb blood flow was evaluated with a Doppler blood flow meter, and as a result, a significant increase in blood flow in the ischemic limb was observed with the administration of peptide (FAMP-5) (FIG. 15).

Macrophage specific RCT was examined. Mice were given intraperitoneal administration of PBS or peptide (FAMP-5) (50 mg / kg) for 5 days. Three days after the start of administration, 3H-cholesterol-labeled macrophages were administered into the abdominal cavity of the mouse, and the amount of 3H-cholesterol in blood and stool was measured. As a result, it was found that fecal excretion of 3H-cholesterol in macrophages was significantly increased by administration of peptide (FAMP-5) (FIG. 16).

Since the cholesterol export peptide according to the present invention has an HDL producing action in addition to the cholesterol export action, it is effective for the prevention and treatment of cardiovascular diseases such as atherosclerosis. From the above, the cholesterol export peptide of the present invention can be applied as a pharmaceutical product either as a single substance, as a peptide-lipid mixture, or as a pharmaceutical composition in combination with other drugs containing these.

Claims

General formula [I]:
H-X1-X2-X3-His-Leu-X4-Thr-Leu-X5-Glu-Lys-Ala-
X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-
X18-OH [I]
[Wherein X1 is Ala, single bond ("-") or general formula [Ia]:
X1e-X1d-X1c-X1b-X1a- [Ia]
(In the formula, X1a means Ala or a single bond ("-"), X1b means Lys, Arg or a single bond ("-"), and X1c means Ala or a single bond ("-"). , X1d means His or a single bond ("-"), and X1e means Tyr or a single bond ("-").)
X2 means Thr, Leu, Lys or Ser,
X3 means Glu, Thr or Asp
X4 means Ser, Phe or Lys
X5 means Ser, Tyr, Trp, Phe or Gly
X6 means a single bond ("-"), Lys, Leu or Arg,
X7 means a single bond ("-"), Pro or Lys,
X8 means a single bond ("-") or Ala,
X9 means a single bond ("-"), Phe or Leu,
X10 means a single bond ("-"), Glu, Gln or Asp,
X11 means single bond ("-") or Asp,
X12 means single bond ("-") or Leu,
X13 means a single bond ("-"), Arg, Leu or Gly,
X14 means a single bond ("-"), Gln or His,
X15 means a single bond ("-"), Gly, Lys or Ser,
X16 means single bond ("-"), Leu or His,
X17 is a single bond ("-"), Leu, Met or general formula [Ib]:
X17a-X17b-X17c-X1d [Ib]
(Where X17a means single bond ("-"), Leu or Met, X17b means Pro, Tyr or single bond ("-"), X17c means Val or single bond ("-") As well as X1d means Leu or a single bond ("-"))
However, when the symbols X1a-X1e and X17a-X17d both mean a single bond ("-"), these single bonds as a whole mean one single bond ("-"), and At the same time, X1 is Ala, X2 is Thr, X3 is Glu, X4 is Ser, X5 is Ser, X6 is Lys, X7 is Pro, X8 is Ala, X9 is Leu, X10 is Glu, X11 is Asp, X12 is Leu, X13 Except Arg, X14 is Gln, X15 is Gly, X16 is Leu, and X17 is Leu. ]
Or a peptide represented by the general formula [II]:
A -X20 (X21-OH)-A [II]
[Wherein A represents the general formula [IIa]:
H-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-
X33-X34-X35-X36-X37-X38-X39-X40-X41-X42-X43-
X44-X45-X46- [IIa]
(Wherein X22 means Ala or Val, X23 means Thr, Leu, Lys or Ser, X24 means Glu, Thr or Asp, X25 means His or Ser, and X26 means Leu or Means Phe, X27 means Ser, Phe or Lys, X28 means Thr or Val, X29 means Leu or Ser, X30 means Ser, Gly, Phe, Tyr or Trp, X31 Means Glu or Leu, X32 means Lys or Ser, X33 means Ala, X34 means Lys, Leu, Arg or single bond ("-"), X35 means Pro, Glu, Lys Or X36 means Ala, Glu or single bond ("-"), X37 means Leu, Tyr or single bond ("-"), X38 means Glu, Gln, Asp, Thr or single bond ("-"), X39 means Asp, Lys or single bond ("-"), X40 means Leu, Lys or single bond ("-") , X41 means Arg, Gly, Leu or a single bond ("-"), X42 means Gln, Leu, Lys, His or a bond ("-"), X43 means Gly, Leu, Lys, Ser or bond ("-") means X44 means Leu, His or bond ("-"))
X20 means an amino acid residue such as Lys, and X21 means an amino acid residue such as Leu or Ala. )
Or a general formula [III]:
(A) 2 -X22 (X23-OH)-(A) 2 [III]
Wherein A has the same meaning as above,
X22 means an amino acid residue such as Lys, and X23 means an amino acid residue such as Leu or Ala. )
The peptide which consists of tetramer represented by these.
The peptide according to claim 1, wherein the peptide has a cholesterol removal action.
The peptide according to claim 1 or 2, wherein the peptide further has an HDL nascent action.
The peptide according to any one of claims 1 to 3, wherein the peptide is represented by any one of peptide numbers 1 to 16.
A pharmaceutical composition comprising the peptide according to claim 1 as an active ingredient.