JP2006016347A - Learning and memory enhancing agent and method for enhancing learning and memory - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a substance efficacious for improving disorders of memory/learning caused by Alzheimer's disease, etc., and having learning and memory enhancing action. <P>SOLUTION: A peptide expressed by: Ala-Pro-Gly-Pro-Arg has the learning and memory enhancing action. Therefore, a functional peptide useful as a learning and memory enhancing agent and a method for enhancing the learning and the memory which uses the peptide are given, respectively. The peptide is the excellent functional peptide, comprises a short amino acid sequence, and contains no particular amino acid, so that the peptide has such advantages as to be prepared at a low cost, to be a raw material having high safety, and to be efficacious when orally administered. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a learning memory accelerator containing a peptide represented by Ala-Pro-Gly-Pro-Arg as an active ingredient. Furthermore, the present invention relates to a method for promoting learning memory using the peptide.

  With the aging of society, Alzheimer's memory / learning disorders are said to triple in the next 30-40 years. Therefore, there is a need for means for treating or preventing such diseases. Under such circumstances, several patent applications have been filed for functional peptides that promote the learning and memory function (Japanese Patent Laid-Open No. 9-12597).

  However, the peptide according to JP-A-9-12597 is a peptide fragment in which the amino acid cysteine at position 16 of the 1-29 amino acid residue fragment of acidic fibroblast growth factor is substituted with another amino acid, and the number of amino acid residues is long. .

  By the way, enterostatin is a five-residue endogenous peptide derived from the amino terminus when spleen procolipase is converted to colipase by trypsin, and has an inhibitory effect on feeding and insulin secretion in rats fed a high-fat diet. It is already known to have. The sequences of rat enterostatin are Val-Pro-Asp-Pro-Arg, Val-Pro-Gly-Pro-Arg and Ala-Pro-Gly-Pro-Arg. On the other hand, human and mouse enterostatin is only Ala-Pro-Gly-Pro-Arg.

  The present inventors examined the effects of the rat enterostatin (Val-Pro-Asp-Pro-Arg) on the living body, and prevention and amelioration of amnesia in which the peptide and its fragment were induced by scopolamine and serum. It has been found that it is effective for lowering cholesterol, and a patent application has been filed for that finding (Japanese Patent Laid-Open No. 2002-80393). In addition, the present inventors have reported that human enterostatin (Ala-Pro-Gly-Pro-Arg) has a cholesterol-lowering action and an anti-analgesic action (Agricultural Chemical Society Annual Meeting 2003 Proceedings).

Japanese Patent Laid-Open No. 9-12597 JP 2002-80393 A Agricultural Chemical Society Annual Report 2003 (March 15, 2003)

  Regarding peptides that enhance learning and memory function, despite the knowledge of Japanese Patent Laid-Open Nos. 9-12597 and 2002-80393, the pathophysiology and expression of Alzheimer's or non-Alzheimer's memory / learning disorders Considering the fact that there are large individual differences in modes, there is a further need for substances having an activity that can improve such pathological conditions. Moreover, considering that it is necessary to take a substance effective for senile diseases for a long period of time, it is desirable that such a substance is highly safe and can be administered orally. Therefore, it is an object of the present invention to provide a learning memory function promoting peptide having such advantages.

  The effect of human enterostatin Ala-Pro-Gly-Pro-Arg (APGPR) on learning memory has not been studied so far. Therefore, as a result of the inventors paying attention to the peptide, it was found that human enterostatin (APGPR) is effective in promoting learning memory. As described above, it is known that the peptide has a serum cholesterol lowering action, and considering that the aging society is progressing rapidly, a learning memory promoting peptide having such a function is provided by the present invention. The significance of what was done is considered to be very large.

  Although rat enterostatin was effective in scopolamine-induced amnestic rats, it did not show learning memory promoting action in normal rats. Since human enterostatin (APGPR) of the present invention promoted learning and memory in normal rats, the effects of human enterostatin of the present invention and rat enterostatin are considered to be essentially different.

  Therefore, the present invention provides a learning memory promoter characterized by containing a peptide represented by APGPR as an active ingredient. Furthermore, the present invention also provides a method for promoting learning memory by administering a peptide represented by APGPR.

  APGPR has an action of promoting learning and memory. Therefore, the present invention provides a functional peptide useful as a learning memory accelerator and a method for promoting learning memory using the peptide. The peptide of the present invention is an excellent functional peptide, and since it has a short amino acid residue and does not contain a special amino acid, it can be prepared at low cost and has the advantage of being effective for oral administration.

  As described above, the present invention relates to a learning memory accelerator comprising a peptide represented by Ala-Pro-Gly-Pro-Arg as an active ingredient, and learning memory by administering the peptide. It provides a method for strengthening. It is possible to develop pharmaceuticals, functional foods, and dietary supplements (so-called supplements) using the peptide learning memory enhancer of the present invention. Here, Ala represents alanine, Pro represents proline, Gly represents glycine, and Arg represents arginine. All of these amino acids are L-forms.

  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 kinds of fragments bisected at an arbitrary position of the peptide bond, and the other A method using an active ester such as 2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) with a raw material having a reactive amino group corresponding to the fragment, or carbodiimide The condensation can be carried out using a method using When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.

  Functional groups that should not participate in the reaction in this reaction step are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (Bz), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting agent include groups capable of forming alkyl esters, benzyl esters and the like. In the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, P- 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, but in the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Furthermore, the peptides of the present invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like.
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 where it uses as a pharmaceutical is demonstrated. The administration route of the peptide used in the present invention may be any of oral administration, parenteral administration, and rectal administration. The learning memory promoting peptide of the present invention can be administered orally or parenterally. The dosage of this peptide varies depending on the type of compound, administration method, patient symptoms, age, etc., but is usually 1 mg / kg to 10 g / kg, preferably 10 mg / kg to 1 g / kg per day. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that 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 are 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, bee gum, titanium oxide, sorbitan fatty acid ester, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polyso Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.

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

  These preparations can contain the peptide of the present invention in a proportion of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other ingredients of therapeutic value.

  In order to produce a solid preparation for oral administration, an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed into a powder, or if necessary, sucrose, Binders such as hydroxypropylcellulose and polyvinylpyrrolidone, disintegrants such as carboxymethylcellulose and carboxymethylcellulose calcium are added, and wet or dry granulation is performed to form granules. In order to produce tablets, these powders and granules may be tableted as they are or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets should be coated with an intestinal solvent base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also. In order to produce capsules, powders or granules are filled into hard capsules, or active ingredients are dissolved as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. Can do.

  In order to produce a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol, and glycerin are dissolved in water to add a transparent syrup, further essential oil, ethanol, etc. to make an elixir, Gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension. These liquid preparations may contain a flavoring agent, a coloring agent, a preservative and the like as desired.

  In order to produce injections, active ingredients such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, pH adjusters, sodium chloride, glucose etc. Dissolve in distilled water for injection with an isotonic agent, filter aseptically and fill into ampules, or add mannitol, dextrin, cyclodextrin, gelatin, etc. . In addition, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like may be added to the active ingredient and emulsified in water to give an emulsion for injection.

  To produce a rectal dosage form, the active ingredient is moistened with a suppository base material such as cacao butter, fatty acid tri, di- and monoglycerides, polyethylene glycol, etc., dissolved, poured into a mold and cooled, or the active ingredient is made of polyethylene. What is necessary is just to coat | cover with a gelatin film | membrane after melt | dissolving in glycol, soybean oil, etc.

  In order to produce an external preparation for skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc., and if necessary, moistened and kneaded to make an ointment, or rosin, alkyl acrylate polymer After being kneaded with an adhesive such as polyalkyl, it is spread on a non-woven fabric such as polyalkyl to obtain a tape.

  Furthermore, it is also an aspect of the present invention that the learning and memory accelerator of the present invention is administered in the form of functional foods or dietary supplements (so-called supplements). It is possible to mix the learning memory enhancer of the present invention in foods and seasonings that are generally eaten or consumed, and such foods and drinks are produced by ordinary methods and are particularly limited. It is not a thing.

  Next, the present invention will be described more specifically with examples. However, the following examples do not limit the scope of the present invention.

(Synthesis of peptides)
Commercially available Fmoc-Arg (Pmc) -Wang resin (substitution rate 0.50 meq / g) 0.60 g was dispensed into a reaction vessel of a PS3 type peptide synthesizer (manufactured by Protein Technologies), and the new peptide was as follows. Synthesis was performed.

  First, the above resin is put in a reaction vessel, and 1 mmol of Fmoc-Pro and 1 mmol of HBTU as an activator dissolved in 10 ml of dimethylformamide containing 0.4 M N-methylmorpholine are used as a reaction vessel. And stirred at room temperature for 20 minutes.

  Fmoc-amino acid was sequentially coupled from the C-terminal in the same manner as in the method of removing Fmoc group in 20 ml of dimethylformamide containing 20% by weight of piperidine, removing the Fmoc group, and coupling Fmoc-Pro as described above. Ala-Pro-Gly-Pro-Arg (Pmc) -resin was obtained. The resin was placed in 10 ml of deprotection solution (82% by volume trifluoroacetic acid, 5% by volume thioanisole, 3% by volume ethanediol, 2% by volume ethylmethyl sulfide, 3% by volume phenol, 5% by volume water) at room temperature. Stir for 4 hours to release the peptide from the resin.

40 ml of cold ether was added thereto to precipitate the peptide, which was further washed 3 times with cold ether to obtain a crude peptide. This was developed and purified by reverse phase chromatography using an ODS column (Cosmosil 5C ₁₈ -AR, 20 × 250 mm) with a linear concentration gradient of acetonitrile containing 0.1% by weight of trifluoroacetic acid, and Ala-Pro-Gly- Pro-Arg was obtained. As a result of analyzing this product with a protein sequencer (type 492A manufactured by Applied Biosystems), it was found to have the above composition.

(Evaluation of learning promotion effect)
The effect of peptides on learning ability was examined by passive avoidance experiments using a step-through device. When a ddy mouse (male, weight 18-20 g) is placed in a light room of a device divided into two light and dark rooms, the mouse enters the dark room because it prefers a dark place. When entering the dark room, current (0.12 mA) is applied from the floor to educate that the dark room is dangerous (training trial). Immediately after the training trial, the peptide of the present invention (Ala-Pro-Gly-Pro-Arg: APGPR) dissolved in ACSF (artificial cerebral spinal fluid) is administered intralaterally at a dose of 3, 10, 30 or 100 nmol / mouse. The test was conducted 24 hours after the training.

That is, the learning ability of the mouse in the presence or absence of the peptide of the present invention was compared by re-inserting the mouse into the same apparatus and measuring the time during which the mouse stayed bright and dark. A mouse administered with ACSF alone was used as a control. The composition of ACSF consists of 138.9 mM NaCl, 3.4 mM KCl, 1.26 mM CaCl ₂ , 4.0 mM NaHCO ₃ , 0.6 mM NaHPO ₄ , 5.6 mM glucose, and pH is 7.4.

  The effect of administering APGPR into the lateral brain (3 nmol / mouse, 10 nmol / mouse, 30 nmol / mouse, 100 nmol / mouse) was examined (FIG. 1). That is, FIG. 1 shows the results of studying the learning promoting action of the above peptide using the time until the mouse placed in the light room of the cage moves to the dark room (step-through latency: sec) as an index. Although the mouse has the property of moving to a dark room, if it learns that the dark room is dangerous by learning, it stays in the bright room. Therefore, if the mouse learns, step-throughlatency becomes long.

  From the results of FIG. 1, it was shown that the step-through latency was prolonged by administering APGPR, and thus the learning ability of mice was promoted by administering the peptide of the present invention. The cut-off value in this experiment was 600 seconds. When the U test was performed by the Mann and Whitney method, a significant difference compared with the control (ACSF) was observed in the group in which APGPR was administered intratracheally at a dose of 10 nmol / mouse (p). <0.05).

  Furthermore, the effect of oral administration of APGPR (100 mg / kg, 300 mg / kg, 500 mg / kg) was examined (FIG. 2). The results of FIG. 2 also show that step-through latency is prolonged in the APGPR administration group, and that the learning ability of mice is enhanced by administering the peptide of the present invention. The cut-off value in this experiment was 600 seconds. When U test was performed by the Mann and Whitney method, a significant difference compared with the control was observed in the group in which APGPR was orally administered at a dose of 300 mg / kg (p <0.05).

  As already mentioned, rat type enterostatin (Val-Pro-Asp-Pro-Arg: VPPDPR) has a cholesterol-lowering action and an anti-amnestic action on mice treated with scopolamine, but a learning memory promoting action on normal mice. Is not shown. In that respect, the action of human enterostatin (APGPR) of the present invention that promotes learning and memory in normal animals is clearly different from rat enterostatin.

  Although data are not shown, the learning and memory promoting action of APGPR was suppressed by administration of lollumide, an antagonist of cholecystokinin. From this result, it is considered that the action of APGPR is mediated by cholecystokinin.

  APGPR has an action of promoting learning and memory. Therefore, the present invention provides a functional peptide useful as a learning memory accelerator and a method for promoting learning memory using the peptide. Since the peptide of the present invention has short amino acid residues and does not contain any special amino acid, it can be prepared at low cost, is a highly safe material, and has an advantage that it is effective by oral administration. . Therefore, the peptides of the present invention have great potential for use as pharmaceuticals, functional foods, and supplements.

FIG. 1 is a graph showing changes in step-through latency by intraventricular administration of the peptide of the present invention. * Represents a significant difference of p <0.05. FIG. 2 is a graph showing changes in step-through latency by oral administration of the peptide of the present invention. * Represents a significant difference of p <0.05.

Claims (5)

A learning memory promoter comprising a peptide represented by Ala-Pro-Gly-Pro-Arg as an active ingredient. A pharmaceutical comprising the learning memory accelerator according to claim 1 as an active ingredient. A functional food containing the learning and memory accelerator according to claim 1 as an active ingredient. A nutritional supplement containing the learning and memory accelerator according to claim 1 as an active ingredient. A method of promoting learning memory using a peptide represented by Ala-Pro-Gly-Pro-Arg.

Images