JP2002080495A - New serum cholesterol lowering peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new functional peptide having a serum cholesterol lowering action. SOLUTION: This new serum cholesterol lowering agent contains a new peptide represented by the formula Leu-X-Arg (X is Ser, Ala or Pro). There has been no report that a tripeptide lowers a serum cholesterol. Since the peptide can be orally administered, the peptide has a high effect.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel orally administrable serum cholesterol lowering agent containing a peptide having a serum cholesterol lowering action.

[0002]

2. Description of the Related Art In recent years, with the westernization of dietary habits in Japan, the tendency of excessive intake of high protein and high fat foods has been increasing, and the incidence of hypercholesterolemia has increased. I have. Also, ischemic heart disease caused by hypercholesterolemia is increasing. It is said that the risk of ischemic heart disease is particularly closely related to LDL cholesterol level. However, it is not necessary to focus solely on LDL cholesterol level.
L cholesterol ratio (= LDL cholesterol value /
HDL cholesterol) and heparin precipitated cholesterol (= LDL cholesterol + VLDL cholesterol) are also evaluated.

[0003] In recent years, attempts have been made to prevent adult diseases with functional peptides. As one of such attempts, the present inventors have disclosed in Japanese Patent Application Laid-Open No. H11-292896 that X-Pr, a derivative of a peptide derived from rice protein, has been disclosed.
o-Leu-Pro-Arg (X is Leu, Ile, M
et, Phe, Trp)
It is reported to have an effect of lowering blood cholesterol. In addition, 4 derived from β-lactoglobin
Β-lactotensin (H
is-Ile-Arg-Leu) has been found to have a cholesterol-lowering effect upon oral administration.
It is also known that a high molecular weight peptide derived from soybean protein having an unknown structure has a cholesterol-lowering effect by adsorbing bile acid and inhibiting its reabsorption.

[0004]

An object of the present invention is to obtain a functional peptide having a cholesterol-lowering effect. By the way, if a functional peptide shorter than a conventionally known peptide is obtained, it is considered that it is unlikely to be degraded in a living body, and there are advantages that the peptide can be easily synthesized. Therefore, it is an object of the present invention to obtain a functional peptide having a cholesterol-lowering effect with a sequence as short as about 3 amino acids.

[0005]

Means for Solving the Problems Accordingly, the present inventors have proposed β
-An ileal contraction peptide derived from κ-casein, which is a milk protein-derived peptide like lactotensin, was examined, considering that it may have a serum cholesterol lowering effect. That is, Casoxin C (Tyr-Ile-P
ro-Ile-Gln-Tyr-Val-Leu-Se
The effect of the r-Arg) fragment peptide on serum cholesterol was examined. Casoxin C binds to the C3a receptor and has ileal contraction activity, as well as immunostimulatory effects and central effects such as anti-analgesic and anti-amnesia. Tyr-V, a fragment peptide of Casoxin C
al-Leu-Ser-Arg is the smallest unit that binds to the C3a receptor like Casoxin C. As a result of examining the physiological activity of a shorter peptide, it was found that a peptide having a structure of Leu-Ser-Arg has an effect of lowering serum cholesterol. The second amino acid is Ala or P
Leu-Ala-Arg, a peptide changed to ro
And Leu-Pro-Arg also had a serum cholesterol lowering effect. Thus, there is no precedent that the tripeptide exhibits a cholesterol lowering effect, which is the most remarkable effect of the present invention.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a Leu-X-Arg
(X is Ser, Ala or Pro) and is a novel peptide. These peptides are useful for lowering serum cholesterol. Therefore, a cholesterol-lowering agent containing one or two of these peptides can be prepared. The peptide of the present invention has 3
Although it is a tripeptide consisting of amino acids, there has never been a precedent in which tripeptide exhibits a cholesterol-lowering effect as in the present invention. As described above, the short sequence of the functional peptide makes it difficult to decompose and facilitates the synthesis. Therefore, the advantage of the tripeptide having a cholesterol-lowering effect is great. Leu mentioned above is leucine, Arg is arginine, Ser is serine, Ala is alanine, and Pro is proline. All such amino acids are in the L-form.

[0007] The peptide of the present invention can be obtained by a peptide synthesis method. That is, in a liquid phase method or a solid phase method, which is a method usually used for peptide synthesis, a raw material having a reactive carboxyl group corresponding to one of two types of fragments bisected at an arbitrary position of a peptide bond, A raw material having a reactive amino group corresponding to the fragment, and 2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethyl
It can be condensed using a method using an active ester such as uronium hexafluorophosphate (HBTU), a method using carbodiimide, or the like. When the resulting condensate has a protecting group, it can be produced also by removing the protecting group.

[0008] In this reaction step, functional groups that should not participate in the reaction are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (B
z), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting agent include groups capable of forming an alkyl ester, a benzyl ester, and the like. In the case of the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, a chloromethyl resin, an oxymethyl resin, It is bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.

After completion of the condensation reaction, the protecting group is removed,
In the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Further, the peptide of the present invention is purified according to a usual method. For example, ion exchange chromatography,
Examples include reversed-phase liquid chromatography and affinity chromatography. The synthesis of the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, or the like.

Next, the case of using as a medicine will be described. The administration route of the peptide used in the present invention includes:
It may be any of oral administration, parenteral administration, and rectal administration.
The serum cholesterol lowering agent of the present invention can be administered orally or parenterally. The dose of the peptide varies depending on the type of the compound, the administration method, the condition of the patient, the age, etc., but is usually 1 mg / kg to 10 g per day.
/ Kg, preferably 10 mg / kg to 1000 mg / k
g. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a preparation carrier. As the pharmaceutical carrier, a substance which is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.

Specifically, examples of such substances include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch,
Carboxymethylcellulose calcium, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan Fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water, etc. No.

Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, and injections.
These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another suitable solvent at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the peptide of the present invention is prepared by dissolving the peptide in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, or may be added with a buffer or a preservative. Good.

These preparations contain the peptide of the present invention in a concentration of 0.1%.
It can be contained at a rate of 01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other components of therapeutic value.

To prepare a solid preparation for oral administration, the active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, and silicic acid anhydride are mixed together to form a powder, or if necessary, Then, a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone and the like, a disintegrant such as carboxymethylcellulose and calcium carboxymethylcellulose are added, and granulated by wet or dry granulation. In order to produce tablets, these powders and granules may be compressed as they are or by adding a lubricant such as magnesium stearate or talc. These granules or tablets should be coated with an enteric base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer and enteric coated, or coated with ethylcellulose, carnauba wax, hydrogenated oil, etc. to form a sustained release preparation. Can also. To produce capsules, powders or granules are filled into hard capsules, or the active ingredient is dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, or the like, and then coated with a gelatin film to form soft capsules. Can be.

To prepare a liquid preparation for oral administration, an active ingredient and a sweetening agent such as sucrose, sorbitol, and glycerin are dissolved in water, and a clear syrup, an essential oil, ethanol and the like are added thereto, and an elixir is added. Alternatively, gum arabic, tragacanth, polysorbate 80, sodium carboxymethylcellulose and the like may be added to form an emulsion or suspension. These liquid preparations may optionally contain a flavoring agent,
You may add a coloring agent, a preservative, etc.

In order to prepare an injection, the active ingredient may be added, if necessary, to a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, Dissolve it in distilled water for injection together with an isotonic agent such as glucose, filter aseptically and fill in ampoules, or add mannitol, dextrin,
Cyclodextrin, gelatin, etc. may be added and freeze-dried in vacuum to obtain a working-solution type injection. Also, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like can be added to the active ingredient and emulsified in water to prepare an emulsion for injection.

To prepare a rectal preparation, the active ingredient is moistened together with a suppository base such as cocoa butter, tri-, di- and monoglycerides of fatty acids, polyethylene glycol, etc., dissolved and poured into a mold, cooled or cooled. The components may be dissolved in polyethylene glycol, soybean oil, or the like, and then coated with a gelatin film.

To prepare an external preparation for skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol and the like, and if necessary, moistened and kneaded into an ointment, or rosin, alkyl acrylate. After kneading with an adhesive such as an ester polymer, it is spread on a non-woven fabric such as a polyalkyl to form a tape.

[0019]

Now, the present invention will be described more specifically below with reference to working examples. (Synthesis of peptide) Commercially available Fmoc-Arg (Pmc)
-Wang resin (substitution rate 0.50 meq / g) 0.60
g to a PS3 peptide synthesizer (Protein Tec)
(made by Hnologies, Inc.), and the novel peptide was synthesized as follows. First, the above resin was placed in a reaction vessel, and 1 mmol of Fmoc-Se
r (tBu) and 1 mmol of HBT as an activator
A solution of U in 10 ml of dimethylformamide containing 0.4 M N-methylmorpholine was added to the reaction tank, and the mixture was stirred and reacted at room temperature for 20 minutes.

The Fmoc group was removed from the obtained resin in 20 ml of dimethylformamide containing 20% by weight of piperidine, and then Fmoc-Leu was coupled in the same manner as in the above-mentioned method of coupling Fmoc-Ser. Leu (Pmc) -Ser-Arg-resin was obtained.
The resin was added at room temperature in 10 ml of deprotection solution (82% trifluoroacetic acid, 5% thioanisole, 3% ethanediol, 2% ethyl methyl sulfide, 3% phenol, 5% water). After stirring for 4 hours, the peptide was released from the resin.

To this was added 40 ml of cold ether to precipitate the peptide, which was further washed three times with cold ether to obtain a crude peptide. This is supplied to an ODS column (Cosmosi
L5C ₁₈ -AR, was obtained by reverse phase chromatography with 20 × 250 mm) developed by linear gradient of acetonitrile containing 0.1 wt% trifluoroacetic acid, purified ,, Leu-Ser-Arg and (LSR) . The product was analyzed using a protein sequencer (Model 477A, manufactured by Applied Biosystems), and as a result, the product was found to have the above composition. In a similar manner, Leu-Ala
-Arg (LAR) and Leu-Pro-Arg (LP
R) was also obtained.

(Measurement of Serum Cholesterol) Male ICR
Mice (20-24 g, 4 weeks old, 8 animals per group) were fed a high cholesterol, high cholic acid diet for 3 days. 0.6% cholesterol and 0.1% for high cholesterol and high cholic acid diets.
It contains 2% cholic acid and the composition of the feed is shown in Table 1. During breeding of the peptide of the present invention for the above three days,
Simultaneously with the start of the experiments on the second and third days, a weight of 50 mg / kg was dissolved in physiological saline and orally administered with a probe. That is, a total of 100 mg / kg was administered. A high cholesterol diet was given even during the administration of the peptide, and after fasting for 24 hours after the second administration of the peptide, blood was collected by cardiac blood sampling.

[0023]

[Table 1]

The collected blood was incubated in a thermostat at 37 ° C. for 30 minutes, centrifuged at 3000 g for 10 minutes, and the serum obtained was assayed for serum cholesterol by an enzyme method. That is, a kit for measuring the concentration of total cholesterol and heparin precipitated cholesterol (Cholesterol E Test Wako and HDL manufactured by Wako Pure Chemical Industries, Ltd.)
(Cholesterol precipitation reagent set) to measure the absorbance at 600 nm. Heparin precipitated lipoprotein (HPL) cholesterol concentration is the sum of LDL cholesterol and VLDL cholesterol, and was obtained by subtracting HDL cholesterol from serum total cholesterol.

The average value of the total cholesterol and heparin precipitated cholesterol concentrations of the test group and the control group to which the peptide of the present invention was administered was determined, and the cholesterol lowering rate (%) was calculated. The reduction rate (%) was expressed as a percentage obtained by subtracting the average value of the test group from the average value of the control group and dividing the value by the average value of the control group. The test group (8
Of the total cholesterol and the heparin precipitated cholesterol concentration (%). The higher the decrease value and the higher the significance level (P), the greater the increase suppression effect.

Leu-Ser-Arg (LSR) and its derivatives Leu-Ala-Arg (LAR) and L
The reduction rate (%) of total cholesterol and heparin precipitated (HPL) cholesterol by administration of eu-Pro-Arg (LPR) was examined by comparison with a control group. Table 2 shows the results. As shown in Table 2, all three peptides of the present invention exhibited a cholesterol lowering rate of 24% or more (n = 8).

[0027]

[Table 2]

From the above results, it was found that the peptide of the present invention was administered to mice administered with a high cholesterol diet at 50 to 100 mg / k.
Oral administration at a dose of g results in a 20-30% serum cholesterol lowering effect. The peptide of the present invention is a safe cholesterol-lowering substance by lowering serum cholesterol in mice administered with a high cholesterol diet by oral administration but not in normal mice.

The mechanism by which the LSR of the present invention exhibits a cholesterol-lowering effect is still unknown. However, among various fragment peptides derived from Casoxin C, T
yr-Val-Leu-Ser-Arg or Val-Le
LSR promoted phagocytosis even though u-Ser-Arg did not show phagocytosis promoting activity. From the above, it is considered that the cholesterol-lowering effect of LSR is due to regulation through the immune system. LSR did not increase the amount of bile acids in feces. From these results, it is considered that the cholesterol lowering effect of LSR is not due to inhibition of bile acid reabsorption.

[0030]

According to the present invention, Leu-X-Arg (X
Was given a novel serum cholesterol lowering agent containing a novel peptide represented by Ser, Ala or Pro). To date, there has been no report that the tripeptide lowers serum cholesterol, and since the peptide of the present invention can be orally administered, the effect of the present invention is great.

Claims (2)

[Claims] 1. Leu-X-Arg (X is Ser, A
la or Pro). 2. Leu-X-Arg (X is Ser, A
a serum cholesterol-lowering agent, comprising a peptide represented by la or Pro) as an active ingredient.