JP4570402B2 - Central function improver - Google Patents

Description

  The present invention relates to a central function improving agent containing a physiologically active peptide represented by Arg-Ile-Tyr, and further relates to a central function improving agent having a feeding-suppressing action and a learning promoting action.

  In recent years, with the progress of an aging society, an increase in lifestyle-related diseases has become a problem, and this is based on obesity after middle-aged and older, and is considered to cause hypertension, diabetes, and arteriosclerosis. The cause of this is thought to be that obesity is caused by a decrease in energy consumption due to lack of exercise, as well as an increase in calories due to excessive food intake, and an imbalance in calorie regulation.

  On the other hand, dementia associated with aging is also a major social problem, and the development of substances that treat or prevent this is desired.

Regarding such dementia, it has been reported that a spinach-derived peptide (Try-Pro-Leu-Asp-Leu-Phe) has a learning promoting action (see, for example, Non-Patent Document 1).
Peptides, vol24, 325-328 (2003)

However, although the peptide described in the above literature has a learning promoting effect, it does not have an effect on feeding suppression.
That is, there is a need for a multi-functional peptide that improves central functions in anticipation of an aging society in the future and also has an antifeedant action and a learning function promoting action.

Therefore, as a result of various studies on peptides to solve such problems, the present inventors have found that the bioactive peptide represented by Arg-Ile-Tyr has a central improvement function that has both an antifeedant action and a learning promoting action. The inventor has completed the present invention.
The inventor has already filed an application for a physiologically active peptide represented by Arg-Ile-Tyr derived from rapeseed (Japanese Patent Laid-Open No. 2004-51636).

  The novel peptide of the present invention is a peptide having an antifeedant action and also a learning function promoting action.

  The peptide having a central function of the present invention is represented by the Arg-Ile-Tyr sequence, where Arg is arginine, Ile is isoleucine, Tyr is tyrosine, and these amino acids are all L-forms. .

The above peptide can be produced by the following peptide synthesis method.
The peptide is synthesized by a liquid phase method or a solid phase method, and in any case, a raw material having a reactive carboxyl group corresponding to one of two kinds of fragments that are 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) or a carbodiimide with a raw material having a reactive amino group corresponding to the fragment Condensation is performed by a conventional method.

  Functional groups that should not participate in the reaction in this condensation 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 protecting group for the carboxyl group include groups capable of forming alkyl esters, benzyl esters, etc. In the case of the solid phase method, the carboxyl group at the C-terminal is chlorotrityl resin, chloromethyl resin, oxymethyl resin, P-alkoxy Bound to a carrier such as benzyl alcohol resin.

After completion of the condensation reaction, the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved, and the product is purified according to the usual method. Examples of such purification include reverse phase liquid chromatography, ion exchange chromatography, affinity chromatography, and the like.
Analysis of the synthesized peptide is performed by a method such as a protein sequencer or LC-MS that reads an amino acid sequence from the C-terminal by Edman degradation.

The central function improving agent of the present invention contains the above-mentioned peptide, and the administration route of the central function improving agent may be any of oral administration, intravascular administration, and rectal administration, but oral administration is preferred. The dose of such a central function improving agent to a person cannot be generally stated depending on the administration method, patient's symptoms and age, etc., but is usually 0.1 to 1000 mg, preferably 1 to 100 mg once per day. It is preferable to set the time.
Such a central function improving agent is usually administered in the form of a preparation. As the carrier and auxiliary agent used in the preparation, a substance that is commonly used in the preparation field and does not react with the peptide is used.

  Specifically, for example, 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, refined lanolin, glycero gelatin, polysorbate, macro Lumpur, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, 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. In the case of a liquid preparation, it may be dissolved or suspended in water or other appropriate medium at the time of use. Tablets and granules may be coated by a known method. In the case of injections, it is prepared by dissolving the central function improving agent in water, but it may be dissolved in physiological saline or glucose solution as necessary, and buffering agents and preservatives may be added. Good.

  These preparations preferably contain the target peptide in an amount of 0.01% by weight or more, more preferably 0.5 to 70% by weight. These formulations may also contain other therapeutically valuable ingredients.

  Next, the present invention will be described more specifically with reference to examples. The test group was expressed as P <0.01 when significant at the significance level of 1% relative to the control group, and P <0.05 when significant at the significance level of 5%.

Example 1

(Peptide synthesis)
0.6 g of commercially available Fmoc-Tyr (tBu) resin (substitution rate: 0.5 meq / g) was dispensed into a reaction vessel of a PS3 type peptide synthesizer (manufactured by Protein Technologies), and synthesis of a new peptide was performed as follows. went.
First, the above resin is put in a reaction vessel, and 1 mmol of Fmoc-Ile and 1 mmol of HBTU as an activator dissolved in 10 ml of dimethylformamide containing 0.4 M N-methylmorpholine are put in a reaction vessel. The mixture was further stirred at room temperature for 20 minutes. The resin was once washed with dimethylformamide and then reacted using Fmoc-Ile and HBTU as described above.
Fmoc-amino acids were sequentially coupled from the C-terminal in the same manner as in the method in which the Fmoc group was removed in 20 ml of dimethylformamide containing 20% by volume of piperidine and then the Fmoc-Ile was coupled. , Arg-Ile-Tyr (tBu) resin was obtained. The resin in 10 ml of deprotection solution (82% by volume trifluoroacetic acid, 5% by volume thioanisole, 3% by volume ethanedithiol, 2% by volume ethylmethyl sulfide, 3% by volume phenol, 5% by volume water). Stir at room temperature for 4 hours to release the peptide from the resin.

40 ml of cold ether was added thereto to precipitate the peptide, and further washed with cold ether three times to obtain a crude peptide. This ODS column _{(Cosmosil 5C 18 -ARII, 20 ×} 250mm) developed by linear gradient of acetonitrile containing 0.1 wt% trifluoroacetic acid by reverse phase chromatography with purified, the Arg-Ile-Tyr A peptide having the sequence was obtained. This product was analyzed by a protein sequencer (“type 492” manufactured by Applied Biosystems) to confirm that the sequence was as described above.

Using the peptides obtained above, the anti-feeding action of Arg-Ile-Tyr was tested by the method shown below.
(Preparation and administration of test compounds)
ddY mice (male, 7 weeks old, Japan SLC Co., Ltd.) were individually raised in cages maintained under certain conditions (22 ° C., light / dark cycle 12 hours), and were allowed to freely feed and feed. Mice were fasted from 24 hours before the test and used for the test. Arg-Ile-Tyr was dissolved in physiological saline and orally administered, and only physiological saline was administered as a control.

(Measurement of food intake)
Arg-Ile-Tyr (30, 50 mg / kg) was orally administered to mice, and the feed weight was measured at 20, 60, 120 and 180 minutes before and after oral administration, and the intake was calculated.

(Statistical analysis)
The results are shown as mean ± standard error. Differences between groups were examined using analysis of variance (ANOVA) with Bonferroni t-test. (P values less than 0.05 were considered significant.)

  As shown in FIG. 1, in the Arg-Ile-Tyr administration group, the intake was suppressed as compared with the control group, and the anti-feeding action of Arg-Ile-Tyr was shown.

(Learning promotion effect)
The effect on learning ability was examined by a passive avoidance experiment using a step-through device. When a ddy mouse (male, weight 24 ± 2 g) is placed in the light room of a device divided into two light and dark rooms, the mouse enters the dark room because it prefers dark places. When entering a dark room, an electric shock (28-29 V, 5 sec duration) is applied from the floor to educate that the dark room is dangerous (training trial). Immediately after the training trial, Arg-Ile-Tyr was administered intracerebrally in a volume of 10 or 30 nmol / mouse, and the test trial was performed 24 hours later. That is, the learning ability of the mouse in the presence or absence of the peptide was compared by placing the mouse in the same apparatus again and measuring the time during which the mouse stayed in the light and dark.

  As shown in FIG. 2, it was shown that administration of Arg-Ile-Tyr prolonged the stay in the bright room, and that administration of the peptide enhanced the learning ability of mice. In FIG. 2, n = 12, and 600 seconds was taken as the cutoff value in this experiment. When U test was performed by the Mann and Whitney method, a significant difference was observed in the group administered with 10 nmol of Arg-Ile-Tyr at a risk rate of 5% compared to the control group (P < 0.05). Moreover, the same tendency was recognized although there was no significant difference in 30 nmol administration.

  The central function-improving agent of the present invention is excellent in an eating inhibitory action and a learning promoting action, and is useful as an eating inhibitor and a learning promoting agent.

Graph showing food intake over time after each administration The graph which shows the residence time in the bright room when Arg-Ile-Tyr is administered into the ventricle at each concentration

Claims (2)

A central function improving agent for feeding suppression, comprising a physiologically active peptide represented by Arg-Ile-Tyr. A central function improving agent for promoting learning, comprising a physiologically active peptide represented by Arg-Ile-Tyr.

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