KR20110035313A - Mimicries of apolipoprotein a-1, and composition containing the same for treating hyperlipemia and disease associated therewith - Google Patents

Abstract

PURPOSE: A therapeutic agent containing an apolipoprotein A-1 mimic peptide for treating hyperlipidemia is provided to ensure the excellent recognition of lipids. CONSTITUTION: An amphipathic peptide library has an amino acid sequence of an amphipathic alpha-helical peptide containing 3-8 phenylalanines at one side. In the amphipathic peptide, an amino acid with a positive charge is substituted with an amino acid containing less carbon than the amino acid. A method for screening an ApoA-1 mimic peptide comprises: a step of preparing the amphipathic peptide library; a step of analyzing the cholesterol release of cells in the amphipathic peptide library; and a step of selecting peptides in which cholesterol release has increased more than ApopA-1. A therapeutic agent for treating hyperlipidemia contains the selected amphipathic peptide as an active ingredient.

Description

Apolipoprotein A-1 mimetic peptide, and hyperlipidemia and related therapeutic agents comprising the same

The present invention relates to an apolipoprotein A-1 (Apolipoprotein A-1, Apo A-I) mimetic peptide, and a hyperlipidemia comprising the same and a therapeutic agent associated with the disease.

Hyperlipemia is a condition in which more fatty substances are present in the blood and accumulated on the walls of blood vessels, causing inflammation and consequently cardiovascular disease. Hyperlipidemia is the normal range of serum lipids when the concentration of one or more serum lipids such as cholesterol, triglycerides, phospholipids and free fatty acids is fasting, triglycerides 50-150 mg / dl, phospholipids 150-250 mg / dl, cholesterol 130 ~ 230 mg / dl and free fatty acids 5 ~ 10 mg / dl. Neglecting hyperlipidemia can lead to serious complications such as hypertension, coronary atherosclerosis (angina, myocardial infarction), and cerebral atherosclerosis (cerebral infarction).

Elevated low density lipoprotein (LDL) is an independent risk factor for hyperlipidemia and is one of the molecules that carries liver or intestinal cholesterol through the blood to tissues. It is reported that it is bad cholesterol, and because it contains a lot of cholesterol, an increase in LDL in the blood may be deposited on the walls of blood vessels, increasing the risk of coronary artery disease and heart attack. Therefore, various drugs for lowering LDL have been developed to treat hyperlipidemia and related diseases. Recently, however, it has been found that raising high density lipoprotein (HDL) in blood rather than lowering LDL is more effective in treating hyperlipidemia.

High density lipoprotein (HDL) is a combination of Apolipoprotein A-1 (apolipoprotein A-1) protein, which is mainly released from macrophages near the capillary or cholesterol esters. Unlike LDL, it takes cholesterol from tissue and flows into veins to be absorbed or degraded by liver cells. Therefore, if this process is continued, the cholesterol concentration of the arterial wall macrophages may be reduced, resulting in thinning of the arterial macrophages and expansion of blood vessels. Eventually, activating 'reverse cholesterol transport' by raising HDL in the blood leads to blood cholesterol lowering effects or cholesterol lowering effects on macrophages. Therefore, the increase in blood HDL concentration is an important factor in the treatment of patients with hyperlipidemia or cardiovascular disease.

To date, two targets for raising HDL are known. One of them, Apo A-I, helps the masses of cholesterol or cholesterol ester units released from macrophages. The Apo A-I protein has a shape having several alpha helices, and stacks the outer portion of an unstable cholesterol mass to form a stable HDL. Therefore, if there is Apo A-I or a compound that mimics it in the blood, HDL may be high in the blood. Two types of peptides mimic the Apo A-I protein and have been proven to be effective in phase 1 or more clinical trials.

Another target protein is cholesterol ester transfer protein (CETP). The protein facilitates the conversion of the resulting HDL to LDL. Therefore, the development of CETP inhibitors that inhibit the function of this protein has been promoted by major pharmaceutical companies such as Pfizer and Merck. However, after Pfizer Pharmaceutical's flagship CETP inhibitor Torcetrapib failed in clinical trials at the end of 2006, concerns about other side effects other than translating HDL into LDL have resulted in CETP's potential as a target for therapeutics that raise HDL. Lost.

Since the failure of CETP inhibitors, Apo AI proteins and mimic peptides thereof have become of interest as targets and drugs that can increase HDL. The two drug candidates that mimic the ApoA-I protein, which have undergone phase 1 clinical trials, are Apo AI _milano protein, which mimics a portion of Apo AI protein, and alpha helix, which is completely different from the amino acid sequence of Apo AI protein. D-4F peptides that are bilateral peptides are known. These compounds are in phase III and phase I respectively at the end of 2007.

The D-4F peptide is being developed for the purpose of oral treatment consisting of 18 D-amino acids, and has a double-sided alpha helix shape. First, in terms of hydrophilicity, lysine is positively charged and glutamic or aspartic acid is negatively charged. Such a peptide may form a macromolecule in which a plurality of peptide molecules are stacked by ionic recognition between positive and negative charges in other peptide molecules when an alpha helix is formed. In other words, the alpha helix peptides form one unit of the fence and are arranged in the shape of the fence as a whole. Molecular recognition proceeds by creating a peptide fence with cholesterol inside. Therefore, the positively and negatively charged residues of the peptide hydrophilic surface are expected to play an important role in the role of the peptide. Although attempts have been made to improve the hydrophilic portion of D-4F, including experiments on relocating positive and negatively charged amino acid residues, no peptide has been reported that exceeds the efficacy of D-4F. Also, in the effort to improve D-4F, improvement using artificial amino acids that do not exist in nature has not been attempted.

On the other hand, the four phenylalanine residues located on the minor side of the D-4F peptide are directly recognizable with cholesterol, so the position and shape of the phenylalanine are important. It is known that the number and location of this phenylalanine is important in 4F (18 amino acids composed of L amino acids) made before D-4F production. It is known to decrease. The role of this phenylalanine is thought to be primarily involved in the recognition of hydrophobic molecules cholesterol or cholesterol esters. However, there is no improvement in the use of more complex aromatic imano acids or the improvement of 4F materials using artificial aromatic amino acids than the extremely simple group called phenylalanine.

Therefore, the present inventors are trying to develop a peptide that can treat hyperlipidemia and related diseases by increasing HDL more effectively than conventional peptides, and using 4F [ApoA- 1) A peptide library was prepared in which the amino acid of the hydrophilic and hydrophobic side of a double-sided alpha helical peptide having four phenylalanine (F) bound to similar lipids was prepared and screened for cholesterol efflux and hydrophobic molecules than 4F. By identifying Apo A-I mimetic peptides with a significantly improved function in the recognition of the present invention, the present invention is confirmed that the present invention can be usefully used as a therapeutic agent for hyperlipidemia and related diseases, which can effectively transport and eliminate cholesterol to liver cells. Completed.

It is an object of the present invention to provide apolipoprotein A-1 (Apolipoprotein A-1, Apo A-1) mimetic peptide, and a therapeutic agent for hyperlipidemia and related diseases comprising the same.

In order to achieve the above object, the present invention, in the amino acid sequence of the amphipathic α-helical peptide having three to eight phenylalanine (phenylalanine, F) on one side, a positively charged amino acid of the hydrophilic amino acid Provided is a bilateral peptide library comprising a bilateral peptide having an amino acid sequence substituted with an amino acid having a lower carbon number than the amino acid and having a negatively charged amino acid with an amino acid having a higher carbon number than the amino acid.

The present invention also relates to an amino acid sequence of an amphipathic α-helical peptide having 3 to 8 phenylalanine (F) on one side, wherein at least one of the hydrophobic amino acids is phenylalanine and the aromatic other than phenylalanine. A bilateral peptide library is provided comprising a bilateral peptide having an amino acid sequence substituted with an amino acid.

In addition,

1) preparing the bilateral peptide library;

2) analyzing the degree of cholesterol outflow of cells of the bilateral peptide library of step 1); And

3) Apolipoprotein A-1 (Apolipoprotein A-1, Apo A-1) provides an Apo A-1 mimetic peptide screening method comprising the step of selecting a peptide that is increased than Apolipoprotein A-1.

Or, the present invention

1) preparing the bilateral peptide library; And

2) Apo A-1 mimetic peptide screening method comprising mixing the tryptophan fluorescent probe molecules in the bilateral peptide library of step 1) and measuring the peptide with an increased tryptophan fluorescence intensity than the Apo A-1 peptide by measuring with a fluorophotometer To provide.

In another aspect, the present invention provides a high density lipoprotein (HDL) enhancer containing the double-sided peptide selected by the method as an active ingredient.

In addition, the present invention provides a therapeutic agent or diagnostic reagent for hyperlipidemia and related diseases containing the double-sided peptide selected by the method as an active ingredient.

Using the Apo A-I mimetic peptide of the present invention, it is possible to screen double-sided peptides for raising high density lipoprotein (HDL) in blood, and peptides in which phenylalanine of the hydrophobic side of selected 4F is substituted with 2-naphthylalanine. Compared to the existing 4F is significantly superior cholesterol outflow and lipid recognition, it can be usefully used as a candidate peptide for Apo A-I mimetic peptides and therapeutic agents for the treatment of hyperlipidemia and related diseases.

Hereinafter, the present invention will be described in detail.

In the amino acid sequence of an amphipathic α-helical peptide having 3 to 8 phenylalanine (F) on one side, a positively charged amino acid among hydrophilic amino acids has a lower carbon number than the amino acid. Provided is a bilateral peptide library comprising a bilateral peptide having an amino acid sequence substituted with an amino acid and a negatively charged amino acid with an amino acid having a higher carbon number than the amino acid.

The bilateral peptide may include, but is not limited to, a peptide consisting of D-amino acids.

In one embodiment of the bilateral peptide library of the present invention, one or two of the hydrophilic amino acids lysine (K) in the amino acid sequence (4F) described in SEQ ID NO: 1 is ornithine (Orn), diaminobutyl Is substituted with diaminobutyric acid (Dab) or diaminopropionic acid (Dap), and one or two of glutamic acid (E) or aspartic acid (D) is glutamic acid or aminoadip It may be a bilateral peptide library including a bilateral peptide having an amino acid sequence substituted with an acid (amino adipic acid, Aad), but is not limited thereto.

The double-sided peptide library preferably comprises any one or more of the peptides having an amino acid sequence set forth in SEQ ID NO: 3 to 24, more preferably comprises SEQ ID NO: 3, but is not limited thereto.

The present invention also relates to an amino acid sequence of an amphipathic α-helical peptide having 3 to 8 phenylalanine (F) on one side, wherein at least one of the hydrophobic amino acids is phenylalanine and the aromatic other than phenylalanine. A bilateral peptide library is provided comprising a bilateral peptide having an amino acid sequence substituted with an amino acid.

The bilateral peptide may include, but is not limited to, a peptide consisting of D-amino acids.

In one embodiment of the bilateral peptide library of the present invention, one or two of the hydrophobic amino acids phenylalanine (F) in the amino acid sequence (4F) described in SEQ ID NO: 1 are alanine (A), tryptophane, W), a bilateral peptide library comprising bilateral peptides having an amino acid sequence substituted with 1-naphthylalanine (Nal1) or 2-naphthylalanine (Nal2).

The bilateral peptide library preferably comprises any one or more of the peptides having an amino acid sequence set forth in SEQ ID NO: 25 to 44, and most preferably comprises SEQ ID NO: 43, but is not limited thereto.

In order to find an Apo A-I mimetic peptide having improved recognition ability with cholesterol while maintaining the alpha helix of the 4F peptide, which is an existing Apo A-I mimetic peptide, while maintaining the alpha helix while maintaining the main amino acid sequence of 4F Peptides were prepared by substituting hydrophilic or hydrophobic amino acid residues.

In the hydrophilic part, a positive charge such as lysine (K) and a negatively charged amino acid such as aspartic acid or glutamic acid form a large number, so they are recognized between peptide molecules by recognition of positive charge-negative charge, and thus these charged amino acids Under the assumption that fine tuning of the cognitive ability can be achieved by controlling the carbon length of, the amine functional groups with positive charges are shortened. Instead, the carbon lengths of the negatively charged acid moieties are increased. . In addition, since two more positively or negatively charged amine functional groups or acid functional groups are more likely to recognize each other, peptides were synthesized by simultaneously changing the carbon number of the two amino acid residues at the closest distance. That is, the hydrophilic amino acid lysine (containing four carbons from lysine, K, and alpha carbon) is derived from ornithine (ornitine, containing three carbons from alpha carbon) and diaminobutyric acid (Dab, alpha carbon). 2 carbons) or diaminopropionic acid (Dap, containing 1 carbon from alpha carbon) or aspartic acid (D) with 1 carbon as compared to glutamic acid (E, aspartic acid) More) or aminoadipic acid (aminoadipic acid, Aad, aspartic acid has two more carbon) (SEQ ID NOS: 3 to 24).

In the hydrophobic part, phenylalanine (F), which is known as one of the large variables of the existing Apo A-I mimetic peptide 4F, was substituted. That is, a double-sided peptide (SEQ ID NO: 25-28) having an amino acid sequence in which 4 phenylalanine (F) on the minority side was replaced with alanine (A), and a peptide in which phenylalanine was replaced with tryptophane (W) ( SEQ ID NO: 29-32), peptide substituted phenylalanine with 1-naphthylalanine (Nal1) (SEQ ID NOs: 33-36), phenylalanine with 2-naphthylalanine (Nal2) A substituted peptide (SEQ ID NO: 37-40) and a peptide in which phenylalanine was substituted with 1-naphthylalanine and 2-naphthylalanine (SEQ ID NOs: 41-44) were prepared. A bilateral peptide library was prepared comprising.

The inventors determined the calculated value of the mass of the Apo A-I mimetic peptide prepared as described above and the actual mass value obtained by the synthesis and purification process (see Table 1). In addition, cholesterol efflux was analyzed for screening of peptides. Cholesterol bound with ³ H radioisotopes was treated in human macrophage lines and each peptide prepared above was treated. By determining the amount of cholesterol released from macrophages by measuring the amount of extracellular and intracellular cholesterol released from macrophages, it is possible to determine the degree of formation of high density lipoprotein (HDL) of the prepared peptide. As a result, the peptide with a change in hydrophilicity showed a similar or relatively high effect compared to 4F (see FIG. 2), and about 150% cholesterol efflux was increased in the case of 1a (SEQ ID NO: 3). On the other hand, peptides with a change in hydrophobicity showed a significant increase compared to peptides with a change in hydrophilicity. In particular, the hydrophobic peptide 2s, in which phenylalanine at positions 3 and 18 were substituted with 2-naphthylalanine, showed an increase in cholesterol efflux activity of about 300% or more compared to the existing 4F. In addition, all four peptides in which phenylalanine was substituted with alanine showed decreased cholesterol efflux, and peptides in which phenylalanine was substituted with tryptophan or naphthylalanine significantly increased the effect of cholesterol efflux (see FIG. 3). Thus, it can be seen that the four phenylalanine residues play an important role in cholesterol outflow, and the hydrophobicity of this position was shown to be the most enhanced when phenylalanine at position 3 was substituted with tryptophan or naphthylalanine. It can be seen that the residue is an important factor in mimicking the Apo A-I protein.

The inventors of the present invention found that the peptides of the hydrophilic or hydrophobic aspect identified through the above screening were either lipids or aqueous solutions of 1a (about 150% increase compared to 4F) or 2s (about 300% increase compared to 4F) with excellent cholesterol efflux effects. In order to determine the degree of recognition ability with the hydrophobic molecule by checking the state, tryptophan fluorescence intensity change was measured using the tryptophan possessed by the peptide. As a result, the 2s peptide was found to increase about 4.3 times in fluorescence intensity in the lipid environment and in the aqueous solution, and 1a was increased by about 2.1 times. It was confirmed that the 2s peptide is superior to the existing 4F peptide (see Figure 4). Thus, it can be seen that the 2s peptide can be strongly recognized with lipids such as cholesterol around.

In order to determine the difference between the peptide and cholesterol cognition according to the cholesterol concentration, the present inventors fabricated a liposome with an increased amount of cholesterol to determine how the fluorescence intensity of the peptide containing tryptophan increases. As a result, the fluorescence intensity was increased as the amount of cholesterol increased as compared to the membrane structure without cholesterol at all, and thus, it was proved that the cholesterol and the peptide had a close mutual recognition (see FIG. 5).

Therefore, by producing an Apo A-I mimetic peptide having a superior effect than the existing Apo A-I protein, it is possible to selectively recognize cholesterol more effectively than the existing 4F to increase HDL in the blood, and thus can be used as a treatment for hyperlipidemia. Therefore, it can be usefully used in place of Apo A-I protein.

The present invention also provides a method for screening ApoA-1 mimicking peptides using the bilateral peptide library of the present invention.

Specifically,

1) preparing the bilateral peptide library;

2) analyzing the degree of cholesterol outflow of cells of the bilateral peptide library of step 1); And

3) ApoA-1 mimetic peptide screening method comprising the step of selecting a peptide with an elevated cholesterol outflow than Apo A-1.

In the above method, the cells of step 1) are preferably macrophages, but not always limited thereto.

In the above method, the degree of cholesterol outflow in step 2) is preferably used, but not limited to, a cholesterol chollux assay.

The present inventors screen the double-sided peptide having a cholesterol level of Apo A-1 peptide or higher, so that the selected double-sided peptide can be usefully used as an Apo A-1 mimetic peptide.

The present invention also provides another method for screening ApoA-1 mimicking peptides using the bilateral peptide library of the present invention.

Specifically,

1) preparing the bilateral peptide library; And

2) ApoA-1 mimetic peptide screening method comprising mixing a tryptophan fluorescent probe molecule to the bilateral peptide library of step 1) and measuring the peptide with an increased tryptophan fluorescence intensity than the Apo A-1 peptide by measuring with a fluorophotometer to provide.

In the above method, the recognition ability with lipid or hydrophobic molecules may be calculated through the tryptophan fluorescence intensity of step 2).

The present inventors screen the bilateral peptides with tryptophan fluorescence intensity greater than or equal to the Apo A-1 peptide, so that the selected bilateral peptides can be usefully used as Apo A-1 mimetic peptides.

In another aspect, the present invention provides a high density lipoprotein (HDL) enhancer containing a double-sided peptide selected by the above method as an active ingredient.

The present invention was prepared by screening Apo A-I mimetic peptides that can increase the high density lipoprotein by changing the hydrophilic or hydrophobic amino acid of the existing double-sided peptide, 2-nitrile of phenylalanine which is an amino acid of 4F hydrophobic side Since alanine-substituted peptides have been shown to have superior cholesterol outflow and lipid recognition ability than 4F, they can be usefully used as HDL enhancers.

The present invention also provides an agent for treating hyperlipidemia or a disease related thereto or a diagnostic reagent containing the double-sided peptide selected by the above method as an active ingredient.

In addition, the present invention provides a reagent for diagnosing hyperlipidemia or a disease associated with the bilateral peptide selected by the method as an active ingredient.

The hyperlipidemia and related diseases are preferably any one selected from the group consisting of hypercholesterolemia, atherosclerosis, coronary artery disease, cardiovascular disease, restenosis, hypertension, angina pectoris, diabetes, obesity, Alzheimer's disease and multiple sclerosis It is not limited.

The therapeutic agent or diagnostic reagent may be used not only the Apo A-I mimetic peptide of the present invention but also peptides modified using D-amino acids.

The present invention was prepared by screening Apo A-I mimetic peptides that can increase the high density lipoprotein by changing the hydrophilic or hydrophobic amino acid of the existing double-sided peptide, 2-nitrile of phenylalanine which is an amino acid of 4F hydrophobic side Since alanine-substituted peptides have been shown to have better cholesterol outflow and lipid recognition than 4F, they may be usefully used as a therapeutic agent or diagnostic reagent for hyperlipidemia or related diseases.

On the other hand, hyperlipidemia comprising the Apo A-I mimetic peptide of the present invention as an active ingredient and a disease treatment agent associated therewith may include 0.0001 to 50% by weight of the active ingredient relative to the total weight of the composition.

The therapeutic agent of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The therapeutic agent of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as needed. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets, such as aqueous solutions, suspensions, emulsions, and the like, which will act specifically on target organs. Target organ specific antibodies or other ligands can be used in combination with the carriers. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The method of administration of the therapeutic agent of the present invention is not particularly limited, but may be parenterally administered (for example, intravenous, subcutaneous, intraperitoneal, topical or nasal) or oral administration according to a desired method. Administration is preferred, and nasal administration is preferred. Dosage ranges depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease. The daily dosage is about 0.005 to 10 mg / kg for the compound, preferably 0.05 to 1 mg / kg, and more preferably administered once to several times a day.

The therapeutic agents of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers.

The present invention also provides a kit for treating hyperlipidemia or a disease associated with the bilateral peptide selected by the method as an active ingredient.

The present invention also provides a kit for diagnosing hyperlipidemia or a disease associated with the bilateral peptide selected by the method as an active ingredient.

The hyperlipidemia and related diseases are preferably any one selected from the group consisting of hypercholesterolemia, atherosclerosis, coronary artery disease, cardiovascular disease, restenosis, hypertension, angina pectoris, diabetes, obesity, Alzheimer's disease and multiple sclerosis It is not limited.

The present invention was prepared by screening Apo A-I mimetic peptides that can increase the high density lipoprotein by changing the hydrophilic or hydrophobic amino acid of the existing double-sided peptide, 2-nitrile of phenylalanine which is an amino acid of 4F hydrophobic side Since alanine-substituted peptides have been shown to have better cholesterol outflow and lipid recognition than 4F, they can be usefully used as components of the treatment and diagnosis kit for hyperlipidemia and related diseases.

The present invention also provides a method for treating hyperlipidemia or a disease associated with the step of administering the therapeutic agent to a subject.

The present invention also provides a method for diagnosing hyperlipidemia or a disease associated with the step of administering the therapeutic agent to a subject.

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably an ape-like animal such as a chimpanzee or gorilla.

The disease associated with hyperlipidemia is preferably any one selected from the group consisting of hypercholesterolemia, atherosclerosis, coronary artery disease, cardiovascular disease, restenosis, hypertension, angina pectoris, diabetes, obesity, Alzheimer's disease and multiple sclerosis I never do that.

The present invention was prepared by screening Apo A-I mimetic peptides that can increase the high density lipoprotein by changing the hydrophilic or hydrophobic amino acid of the existing double-sided peptide, 2-nitrile of phenylalanine which is an amino acid of 4F hydrophobic side Since alanine-substituted peptides have been shown to have better cholesterol outflow and lipid recognition than 4F, they may be useful for the treatment and diagnosis of hyperlipidemia and related diseases.

The method of administration may be administered orally or parenterally, and may be administered by intravenous, intraperitoneal, subcutaneous, intraperitoneal, rectal, intracerebroventricular or intrathoracic injection. Dosage ranges depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease. The daily dosage is about 0.005 to 10 mg / kg for the compound, preferably 0.05 to 1 mg / kg, and more preferably administered once to several times a day.

In addition, in the amino acid sequence of an amphipathic α-helical peptide having four phenylalanine (F), a positively charged amino acid among hydrophilic amino acids is replaced with an amino acid having a lower carbon number than the amino acid. The present invention provides a use of a bilateral peptide having an amino acid sequence in which a negatively charged amino acid is substituted with an amino acid having a higher carbon number than that of the amino acid in the manufacture of a therapeutic agent or diagnostic reagent for hyperlipidemia or a disease related thereto.

The present invention also relates to an amino acid sequence of an amphipathic α-helical peptide having four phenylalanine (F), wherein at least one of the hydrophobic amino acids is substituted with an aromatic amino acid other than phenylalanine. Provided is a use of a bilateral peptide having a sequence for the manufacture of a therapeutic agent or diagnostic reagent for hyperlipidemia or a disease associated therewith.

The hyperlipidemia and related diseases are preferably any one selected from the group consisting of hypercholesterolemia, atherosclerosis, coronary artery disease, cardiovascular disease, restenosis, hypertension, angina pectoris, diabetes, obesity, Alzheimer's disease and multiple sclerosis It is not limited.

The present invention was prepared by screening Apo A-I mimetic peptides that can increase the high density lipoprotein by changing the hydrophilic or hydrophobic amino acid of the existing double-sided peptide, 2-nitrile of phenylalanine which is an amino acid of 4F hydrophobic side Since alanine-substituted peptides were found to have better cholesterol outflow and lipid recognition than 4F, Apo A-I mimetic peptides could be usefully used as an active ingredient for treating hyperlipidemia or related diseases.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

Example 1 Synthesis of Peptide Library

<1-1> Synthesis of Peptides

The synthesis of peptides was carried out according to the well-known Fmoc solid phase peptide synthesis method. Rinse amide resin (Novabiochem) 50 mg (0.064 mmol) in a container, 1 ml of methylene chloride inflated for 5 minutes, and then dimethylformamide (DMF) 1 ㎖ was added and inflated for 5 minutes. Resin was deprotected three times for 5 minutes with 1 ml of 20% piperidine dissolved in DMF and then washed 5 times with 1 ml of DMF. 6 equivalents of Fmoc deprotected amino acid, (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate [(benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate, PyBop] and diisopropylethylamine ( The solution in which 6 equivalents (198 mg) of diisopropylethylamine and DIPEA) was added was reacted for 60 minutes or longer. After the reaction was completed, it was washed three times with 1 ml of DMF. The presence of the reaction was confirmed by 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) test. That is, one drop of 10% DIPEA dissolved in DMF and one drop of TNBS dissolved in DMF were dropped onto the sample, and the reaction was completed if it was colorless. When one amide binding reaction was completed by the above method, 18-mer peptides were synthesized by repeating the above procedure 18 times using sequentially Fmoc-amino acids conforming to the planned sequence. After deprotection of the synthesized peptide, 90% DMF and 10% methylene chloride were added with 6 equivalents of acetic anhydride and N-hydroxybenzotriazole (HOBt), respectively. 1 ml of methylene chloride) solution was reacted for at least 45 minutes to attach an acetyl group to the N-terminus of the peptide. After acetylation of the N-terminus of the peptide, the resin was washed three times with 1 ml of DMF and 1 ml of methanol and dried in vacuo. 50 mg of the peptide-binding resin synthesized by the solid phase peptide synthesis method was prepared by cleavage solution [2.5% triisopropylsilane (TIS), 2.5% water, 95% trifluoroacetic acid (TFA). )] Was added to 1 ml and stirred for 2 hours to separate the synthesized peptide from the resin. The resin was filtered from the resultant, excess TFA was removed using nitrogen, and 50 ml of n-hexane: diethyl ether (V: V = 1: 1) solution, which had been cooled to 0 ° C., was added. Synthesized peptide was eluted.

<1-2> Purification of Peptides

The peptide eluted in Example <1-1> was dissolved in dimethyl sulfoxide and purified by HPLC using a C18 column. As the HPLC solvent, water and acetonitrile added with 0.1% TFA were used. More specifically, the crude peptide was dissolved in dimethyl sulfoxide at a concentration of about 10 mg / ml and injected into the HPLC 100 μl at a time, and the composition of the solvent was changed for 40 minutes from 5% acetonitrile to 70% acetonitrile. The peptide was purified. At this time, the flow rate was 3 ml / min, the detection wavelength was 220 nm, and the peptide was separated and recovered in 20 to 30 minutes, and then decompressed to evaporate acetonitrile and freeze-dried. The acridine-alpha helical peptide conjugate purified by the above method was identified by measuring the molecular weight by MOLDI-TOF mass spectrometry.

As a result, the sequence of the prepared peptide, the calculated value of the mass and the actual measured value obtained by the synthesis and purification were as shown in Table 1 below.

SEQ ID NO:
A short name Amino acid sequence Mass (M + H ⁺ ) Calculated Value Measures One 4F Ac-DWFKAFYDKVAEKFKEAF 2310 2312.2 2 Sc-4F Ac-DWFAKDYFKKAFVEEFAK 2309.1 2311.2 3 1a Ac-EWFKAFYDKVAEKFKEAF 2323.1 2324.2 4 1b Ac-AadWFKAFYDKVAEKFKEAF 2337.2 2339.9 5 1c Ac-DWFOrnAFYDKVAEKFKEAF 2295.1 2397 6 1d Ac-DWFDabAFYDKVAEKFKEAF 2281.1 2282.1 7 1e Ac-DWFDapAFYDKVAEKFKEAF 2267.1 2268 8 1f Ac-EWFKAFYEKVAEKFKEAF 2337.2 2338.8 9 1 g Ac-EWFKAFYAadKVAEKFKEAF 2351.2 2353.1 10 1h Ac-EWFKAFYDOrnVAEKFKEAF 2309.1 2310.9 11 1i Ac-EWFKAFYDDabVAEKFKEAF 2295.1 2297 12 1j Ac-EWFKAFYDDapVAEKFKEAF 2281.1 2282.9 13 1k Ac-EWFKAFYDKVADKFKEAF 2309.1 2309.5 14 1l Ac-EWFKAFYDKVAAadKFKEAF 2337.1 2337.2 15 1m Ac-EWFKAFYDKVAEOrnFKEAF 2309.1 2310.6 16 1n Ac-EWFKAFYDKVAEDabFKEAF 2295.1 2295.4 17 1o Ac-EWFKAFYDKVAEDapFKEAF 2281.1 2281.3 18 1p Ac-EWFKAFYDKVAEKFKDAF 2309.1 2310.3 19 1q Ac-EWFKAFYDKVAEKFKAadAF 2337.2 2337.9 20 1r Ac-EWFKAFYDKVAEKFOrnEAF 2309.1 2310.4 21 1 s Ac-EWFKAFYDKVAEKFDabEAF 2295.1 2298.8 22 1t Ac-EWFKAFYDKVAEKFDapEAF 2281.1 2281.9 23 1u Ac-EWFDabAFYDKVAEKFKEAF 2295.1 2296 24 1v Ac-EWFDabAFYDKVAEKFDabEAF 2281.1 2281.9 25 2a Ac-EWAKAFYDKVAEKFKEAF 2247.1 2247.9 26 2b Ac-EWFKAAYDKVAEKFKEAF 2247.1 2247 27 2c Ac-EWFKAFYDKVAEKAKEAF 2247.1 2247.9 28 2d Ac-EWFKAFYDKVAEKFKEAA 2247.1 2246.9 29 2e Ac-EWWKAFYDKVAEKFKEAF 2362.1 2363.4 30 2f Ac-EWFKAWYDKVAEKFKEAF 2362.1 2363.3 31 2 g Ac-EWFKAFYDKVAEKWKEAF 2362.1 2363.4 32 2h Ac-EWFKAFYDKVAEKFKEAW 2362.1 2363.4 33 2i Ac-EWNal ₁ KAFYDKVAEKFKEAF 2373.2 2374.4 34 2j Ac-EWFKANal ₁ YDKVAEKFKEAF 2373.2 2374.3 35 2k Ac-EWFKAFYDKVAEKNal ₁ KEAF 2373.2 2374.6 36 2l Ac-EWFKAFYDKVAEKFKEANal ₁ 2373.2 2374.4 37 2 m Ac-EWNal ₂ KAFYDKVAEKFKEAF 2373.2 2374.4 38 2n Ac-EWFKANal ₂ YDKVAEKFKEAF 2373.2 2374.3 39 2o Ac-EWFKAFYDKVAEKNal ₂ KEAF 2373.2 2374.6 40 2p Ac-EWFKAFYDKVAEKFKEANal ₂ 2373.2 2374.4 41 2q Ac-EWNal ₁ KAFYDKVAEKFKEANal ₂ 2423.2 2423 42 2r Ac-EWFKANal ₁ YDKVAEKFKEANal ₂ 2423.2 2423 43 2s Ac-EWNal ₂ KAFYDKVAEKFKEANal ₂ 2423.2 2423 44 2t Ac-EWFKANal ₂ YDKVAEKFKEANal ₂ 2423.2 2423

Example 2 Confirmation of Cholesterol Leakage of Apo A-I Mimic Peptides

In order to screen which compounds of the peptides prepared in <Example 1> mimic Apo A-I well, the peptide was treated with a macrophage line and a cholesterol efflux assay was performed. Specifically, DMEM (Dulbecco's Modified Eagle medium) medium containing penicillin-streptomycin and 10% fetal bovine serum (FBS) was used as a cell culture medium. Macrophages were incubated in a 5% CO ₂ incubator at 37 ° C. for 24 hours by dispensing 5 * 10 ⁴ cells per well in 24-well culture dishes. DMEM (Dulbecco's Modified Eagle medium) supplemented with penicillin-streptomycin and 10% Lipoprotein deficient serum (LPDS) was prepared as a replacement culture to remove all existing cell cultures from each well. , 0.3 ml of each of the replacement cultures were incubated in a 5% CO ₂ incubator at 37 ° C. for 12-16 hours. After incubation, the culture solution of each well was removed, and macrophages were washed by adding 1X phosphate buffered saline (PBS) and shaking. Then, ³ H-cholesterol ⁽³ H-labeled cholesterol) attached label by replacing culture 1 μCi / ㎖ and Ac-LDL 50 ㎍ / to a well-mixed culture liquid into ㎖ 5% of putting 37 ℃ by 0.3 ㎖ to each well Cultured in a CO ₂ incubator. After 24 hours of incubation, the culture medium was removed, the cells were washed three times with 1X PBS, and then incubated for 1 hour in 1% bovine serum albumin (BSA). After incubation, 20 μM of non-cytotoxic peptides were dissolved in DMEM medium containing penicillin-streptomycin, and then 0.3 ml of each well was incubated for 4 hours. At this time, the peptide (Sc-4F) synthesized upside down the amino acid sequence of 4F as a control of 4F was used as a negative control. Thereafter, the culture medium and the cells were separated, and radioactivity was measured using a liquid scintillation counter. The degree of cholesterol outflow was expressed as the ratio of the radioactivity of cholesterol present in the cells and the culture medium, respectively.

As a result, as shown in FIG. 2, the peptide (SEQ ID NOs: 3 to 24) that changed the hydrophilicity treated at 20 μM concentration showed a not so great effect as compared to 4F. Among the peptides that changed the hydrophilicity, the most effective was the 1a (SEQ ID NO: 3) peptide, which was measured to increase cholesterol efflux by 150% compared to the existing 4F, but most peptides showed a similar effect to 4F (FIG. 2). On the other hand, as shown in FIG. 3, the peptide (SEQ ID NOs: 25 to 44) that changed the hydrophobic side treated at 20 μM concentration showed a relatively significant change compared to the peptide that changed the hydrophilic side. All four peptides that substituted phenylalanine with alanine showed a significant decrease in cholesterol efflux effect. Compared to 4F, 2s, which is a peptide substituted with 2-naphthylalanine of a rather large size compared to phenylalanine, has been shown to enhance cholesterol efflux effect by about 300%. Positionally, when the phenylalanine at position 3 was substituted with tryptophan or 2-naphthylalanine, a relatively significant effect was shown (FIG. 3).

Example 3 Recognition of Apo A-I Mimic Peptides and Hydrophobic Molecules

<3-1> Fabrication of Large Unilamellar Vesicles (LUVs)

In a total of 10 mg lipid mixture of phosphatidylcholine (soybean) and cholesterol so that the cholesterol content of LUVs is 0%, 10%, 20%, 30% and 40%, respectively, the volume of chloroform and methanol is 2: 1. 2 mL of the mixed solution was dissolved. After the solvent was removed using a distillation concentrator, 1 mL of tertiary distilled water was hydrated in the thin lipid membrane remaining in the flask. The solution was passed five times through a 0.2 μm polycarbonate filter paper and five times through a 0.1 μm polycarbonate filter paper in a cylindrical extruder to produce a liposome of the same size. The concentration of cholesterol contained in the prepared lyposomes was measured using a cholesterol Lab-assay kit.

<3-2> tryptophane fluorescence measurement

Fluorescence emission spectra were measured at 20 ° C. using an AMINCO-Bowman Series Luminescence Spectrometer. The buffer solution was 140 mM sodium chloride (NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (N-2-Hydroxyethylpiperazine- N'-2-Ethanesulfonic Acid, HEPES, pH 7.4) was prepared. Peptides were maintained at 0 ° C. at 500 μM concentration. 1 μl of the peptide was added to 499 μl of the buffer solution, and the fluorescence emission spectrum was measured at a final concentration of 1 μM. After adding the final concentration of the liposome to the buffer solution and the peptide mixture under the above conditions so that the final concentration of 0.1 mg / ㎖, the spectrum was measured to determine the ratio of the intensity (intensity) when and without the It was.

As a result, as shown in FIG. 4, tryptophan fluorescence intensity was about 1.9 and about 2.1 in the case of 4F and 1a, but 2s reached about 4.3 under the large monolayer vesicle state. Compared to 4F or hydrophilic peptide 1a, the hydrophobic peptide 2s significantly increased the fluorescence intensity of tryptophan, but also showed a rapid shift in wavelength (Fig. 4).

In addition, as shown in Figure 5, as a result of measuring the tryptophan fluorescence intensity using a liposome prepared by increasing the relative amount of cholesterol, 4F and sc-4F shows a fluorescence intensity irrespective of the concentration of cholesterol, In the case of the 2s peptide, the fluorescence intensity was markedly increased by the increase in the concentration of cholesterol in the liposome, and thus, the 2s peptide was recognized to have a high recognition ability with cholesterol (FIG. 5).

As described above, since the Apo A-I mimetic peptide of the present invention exhibits an excellent therapeutic effect on hyperlipidemia compared to the existing peptide, it can be usefully used as a candidate peptide for the development and production of therapeutic agents for hyperlipidemia and related diseases. have.

The new drug that raises HDL among hyperlipidemia drugs is facing a serious crisis due to the failure of CETP inhibitors. Since CETP inhibitors play an important role besides converting HDL to LDL, CETP and its inhibitors are unlikely to be used as targets and target inhibitors to increase HDL unless solutions to this important role are available. .

Then the only remaining target at present is a protein or peptide that mimics Apo A-I. Since D-4F, a precursor of the present invention, is also in a phase 1 clinical trial due to various problems, the preparation of second and third generation Apo A-I mimetic peptides that enhance the efficacy of D-4F is timely. Thus, the new peptides presented in the present invention are suitable as a therapeutic agent for hyperlipidemia because at least from the biochemical or biophysical point of view, they may have a 200-300% superior therapeutic effect compared to existing peptides.

1 is a diagram showing a wheel diagram of the 4F used as a parent of the present invention.

FIG. 2 is a graph showing cholesterol eflux results of a series of peptides with altered residues on the hydrophilic side of the double-sided peptide 4F (each peptide used at 20 μM concentration).

FIG. 3 is a graph showing cholesterol efflux results of a series of peptides varying the residues of the hydrophobic side of the bilateral peptide 4F (each peptide is used at 20 μM concentration).

4 is a graph showing tryptophan fluorescence intensity of selected peptides under lipid environment (Large Unilamella Versicles, LUV). Black indicates the fluorescence intensity when the peptide is in solution, gray indicates the fluorescence intensity when the peptide is in a lipid environment (each peptide uses 1 μM and liposomes at a concentration of 0.1 mg / ml). ).

Figure 5 is a graph showing the change in fluorescence intensity according to the cholesterol composition of the large monolayer vesicles.

<110> SEOUL NATIONAL UNIVERSITY R & DB Foundation <120> Mimicries of Apolipoprotein A-1, and composition containing the          same for treating hyperlipemia and disease associated therewith <130> 9p-09-65 <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 4F <400> 1 Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Sc-4F <400> 2 Asp Trp Phe Ala Lys Asp Tyr Phe Lys Lys Ala Phe Val Glu Glu Phe   1 5 10 15 Ala lys         <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1a <400> 3 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1b <220> <221> VARIANT <222> (1) <223> X is Aad <400> 4 Xaa Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 5 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1c <220> <221> VARIANT <222> (4) <223> X is Orn <400> 5 Asp Trp Phe Xaa Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1d <220> <221> VARIANT <222> (4) <223> X is Dab <400> 6 Asp Trp Phe Xaa Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1e <220> <221> VARIANT <222> (4) <223> X is Dap <400> 7 Asp Trp Phe Xaa Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1f <400> 8 Glu Trp Phe Lys Ala Phe Tyr Glu Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 9 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1 g <220> <221> VARIANT <222> (8) <223> X is Aad <400> 9 Glu Trp Phe Lys Ala Phe Tyr Xaa Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1h <220> <221> VARIANT <222> (9) <223> X is Orn <400> 10 Glu Trp Phe Lys Ala Phe Tyr Asp Xaa Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1i <220> <221> VARIANT <222> (9) <223> X is Dab <400> 11 Glu Trp Phe Lys Ala Phe Tyr Asp Xaa Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1j <220> <221> VARIANT <222> (9) <223> X is Dap <400> 12 Glu Trp Phe Lys Ala Phe Tyr Asp Xaa Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 13 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1k <400> 13 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Asp Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 14 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1 l <220> <221> VARIANT <222> (12) <223> X is Aad <400> 14 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Xaa Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 15 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1m <220> <221> VARIANT <222> (13) <223> X is Orn <400> 15 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Xaa Phe Lys Glu   1 5 10 15 Ala phe         <210> 16 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1n <220> <221> VARIANT <222> (13) <223> X is Dab <400> 16 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Xaa Phe Lys Glu   1 5 10 15 Ala phe         <210> 17 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1o <220> <221> VARIANT <222> (13) <223> X is Dap <400> 17 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Xaa Phe Lys Glu   1 5 10 15 Ala phe         <210> 18 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1p <400> 18 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Asp   1 5 10 15 Ala phe         <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1q <220> <221> VARIANT <222> (16) <223> X is Aad <400> 19 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Xaa   1 5 10 15 Ala phe         <210> 20 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1r <220> <221> VARIANT <222> (15) <223> X is Orn <400> 20 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Xaa Glu   1 5 10 15 Ala phe         <210> 21 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1s <220> <221> VARIANT <222> (15) <223> X is Dab <400> 21 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Xaa Glu   1 5 10 15 Ala phe         <210> 22 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1t <220> <221> VARIANT <222> (15) <223> X is Dap <400> 22 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Xaa Glu   1 5 10 15 Ala phe         <210> 23 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 1u <400> 23 Glu Trp Phe Asp Ala Asx Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe   1 5 10 15 Lys Glu Ala Phe              20 <210> 24 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 1v <220> <221> VARIANT <222> (4) <223> X is Dab <220> <221> VARIANT <222> (15) <223> X is Dab <400> 24 Glu Trp Phe Xaa Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Xaa Glu   1 5 10 15 Ala phe         <210> 25 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2a <400> 25 Glu Trp Ala Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 26 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2b <400> 26 Glu Trp Phe Lys Ala Ala Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 27 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2c <400> 27 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Ala Lys Glu   1 5 10 15 Ala phe         <210> 28 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2d <400> 28 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Ala         <210> 29 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2e <400> 29 Glu Trp Trp Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 30 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2f <400> 30 Glu Trp Phe Lys Ala Trp Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 31 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2 g <400> 31 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Trp Lys Glu   1 5 10 15 Ala phe         <210> 32 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2h <400> 32 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Trp         <210> 33 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2i <220> <221> VARIANT <222> (3) <223> X is Nal1 <400> 33 Glu Trp Xaa Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 34 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2j <220> <221> VARIANT <222> (6) <223> X is Nal1 <400> 34 Glu Trp Phe Lys Ala Xaa Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 35 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2k <220> <221> VARIANT <222> (14) <223> X is Nal1 <400> 35 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Xaa Lys Glu   1 5 10 15 Ala phe         <210> 36 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2l <220> <221> VARIANT <222> (18) <223> X is Nal1 <400> 36 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Xaa         <210> 37 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2m <220> <221> VARIANT <222> (3) <223> X is Nal2 <400> 37 Glu Trp Xaa Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 38 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2n <220> <221> VARIANT <222> (6) <223> X is Nal2 <400> 38 Glu Trp Phe Lys Ala Xaa Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala phe         <210> 39 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2o <220> <221> VARIANT <222> (14) <223> X is Nal2 <400> 39 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Xaa Lys Glu   1 5 10 15 Ala phe         <210> 40 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2p <220> <221> VARIANT <222> (18) <223> X is Nal2 <400> 40 Glu Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Xaa         <210> 41 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2q <220> <221> VARIANT <222> (3) <223> X is Nal1 <220> <221> VARIANT <222> (18) <223> X is Nal2 <400> 41 Glu Trp Xaa Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Xaa         <210> 42 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2r <220> <221> VARIANT <222> (6) <223> X is Nal1 <220> <221> VARIANT <222> (18) <223> X is Nal2 <400> 42 Glu Trp Phe Lys Ala Xaa Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Xaa         <210> 43 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2s <220> <221> VARIANT <222> (3) <223> X is Nal2 <220> <221> VARIANT <222> (18) <223> X is Nal2 <400> 43 Glu Trp Xaa Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Xaa         <210> 44 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> 2t <220> <221> VARIANT <222> (6) <223> X is Nal2 <220> <221> VARIANT <222> (18) <223> X is Nal2 <400> 44 Glu Trp Phe Lys Ala Xaa Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu   1 5 10 15 Ala Xaa          

Claims (17)

In the amino acid sequence of an amphipathic α-helical peptide having 3 to 8 phenylalanine (F) on one side, a positively charged amino acid among hydrophilic amino acids is replaced with an amino acid having a lower carbon number than the amino acid. And a double-sided peptide library having a double-sided peptide having an amino acid sequence in which a negatively charged amino acid is substituted with an amino acid having more carbon atoms than the amino acid. The bilateral peptide library according to claim 1, wherein the substituted positively charged amino acid has an amine group, and the substituted negatively charged amino acid has an acid functional group. The method of claim 1, wherein one or two of the lysine (K) hydrophilic amino acids in the amino acid sequence represented by SEQ ID NO: 1 is ornithine (Orn), diaminobutyric acid (Dab) or dia Substituted with diaminopropionic acid (Dap) and one or two of glutamic acid (E) or aspartic acid (D) substituted with glutamic acid or amino adipic acid (Aad) Double-sided peptide library comprising a double-sided peptide having an amino acid sequence. The bilateral peptide library according to claim 3, wherein the bilateral peptide comprises any one or more of peptides having an amino acid sequence set forth in SEQ ID NOs: 3 to 24. In the amino acid sequence of an amphipathic α-helical peptide having 3 to 8 phenylalanine (F) on one side, an amino acid in which at least one of the hydrophobic amino acids is substituted by an aromatic amino acid other than phenylalanine. A bilateral peptide library comprising bilateral peptides having sequences. 6. The bilateral peptide library according to claim 5, wherein the aromatic amino acid other than phenylalanine is any one selected from the group consisting of phenylalanine derivatives, tryptophan and derivatives thereof, and naphthylalanine and derivatives thereof. The method according to claim 5, wherein one or two of the hydrophobic amino acids phenylalanine (F) in the amino acid sequence represented by SEQ ID NO: 1 are alanine (A), tryptophane (W), 1-naphthylalanine ( A bilateral peptide library comprising a bilateral peptide having an amino acid sequence substituted with 1-naphthylalanine, Nal1) or 2-naphthylalanine, Nal2. The bilateral peptide library according to claim 7, wherein the bilateral peptide comprises any one or more of peptides having an amino acid sequence set forth in SEQ ID NOs: 25-44. 1) preparing the bilateral peptide library of claim 1 or 5; 2) analyzing the cholesterol leakage of the cells of the bilateral peptide library; And 3) ApoA-1 mimetic peptide screening method comprising the step of selecting a peptide with an elevated cholesterol outflow than ApoA-1. 10. The method according to claim 9, wherein the cells of step 1) are macrophages. 10. The method according to claim 9, wherein the cholesterol efflux of step 2) is ApoA-1 mimetic peptide screening method, characterized in that using a cholesterol ester flux (cholesterol efflux assay). 1) preparing the bilateral peptide library of claim 1 or 5; And 2) ApoA-1 mimetic peptide screening method comprising mixing a tryptophan fluorescence probe molecule to the bilateral peptide library and measuring the peptide with increased tryptophan fluorescence intensity than ApoA-1 by measuring with a fluorescence photometer. A high density lipoprotein (HDL) enhancer containing the double-sided peptide selected by the method of claim 9 or 12 as an active ingredient. A therapeutic agent or diagnostic reagent for hyperlipidemia or a disease associated with the bilateral peptide selected by the method of claim 9 or 12 as an active ingredient. 15. The method of claim 14, wherein the hyperlipidemia and related diseases are selected from the group consisting of hypercholesterolemia, atherosclerosis, coronary artery disease, cardiovascular disease, restenosis, hypertension, angina pectoris, diabetes, obesity, Alzheimer's disease and multiple sclerosis Hyperlipidemia and related diseases, therapeutic agents or diagnostic reagents, characterized in that one. A method of treating hyperlipidemia and related diseases comprising administering to a subject the therapeutic agent of claim 14. 15. A method for diagnosing hyperlipidemia and related diseases, comprising administering the diagnostic reagent of claim 14 to an individual.

Images