JP2002080393A - Antiamnestic peptide and serum cholesterol depressing peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a functional peptide effective for senile diseases. SOLUTION: The invention provides a functional peptide useful as a preventing agent or ameliorating agent for amnesia based on the finding that enterostatin (Val-Pro-Asp-Pro-Arg) and its fragment peptide (Pro-Asp-Pro-Arg, Asp-Pro-Arg and cyclo(Asp-Pro)) have antiamnestic action and further provide a functional peptide useful as a serum cholesterol depressing agent based on the finding that the fragment peptide (Pro-Asp-Pro-Arg and Asp-Pro-Arg) of enterostatin has a serum-cholesterol depressing action.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a functional peptide useful as a preventive or ameliorative agent for amnesia. Further, the present invention relates to a functional peptide useful as a serum cholesterol lowering agent.

[0002]

2. Description of the Related Art In recent years, with the advent of an aging society, there is a demand for treating or preventing senile diseases such as senile dementia and cardiovascular diseases caused by hypercholesterolemia. Conventionally, as substances having an anti-amnesic effect, peptides having opioid activity such as analgesia and anesthesia (JP-A-9-227590), prolyl proline derivatives (JP-A-8-3132), strept having prolyl endopeptidase inhibitory activity FERMENTAL PRODUCT BU-4
164EA and B (JP-A-6-279413), enduracidin A having a prolyl endopeptidase inhibitory action (JP-A-5-301826), acetylcholinesterase inhibitory activity, and activity against scopolamine-induced amnesia model Quinoline derivatives (Japanese Unexamined Patent Publication No.
79355), and a peptide SNA-115 (JP-A-5-262795), which has a prolyl endopeptidase inhibitory activity and is a fermentation product of a microorganism of the genus Microbisbora, is known.

[0003] On the other hand, there have been many reports on functional peptides useful for the prevention or treatment of various diseases in recent years, and the present inventors have also reported that Gly-Tyr-Pro-Met-T.
It has been reported that orizatensin, a functional peptide contained in rice protein represented by yr-Pro-Leu-Pro-Arg, has anti-opioid activity, phagocytosis activity, and interleukin 1 production promoting activity (JP-A-Hei. 7-258288). In addition, the present inventors have found that Tyr, which is obtained from a tryptic digest of milk casein (κ-casein) and exhibits ileal contractile activity and immunostimulatory activity via complement C3a receptor, is known as an anti-opioid peptide, Tyr. -Ile-Pro-Ile-Gln-Ty
It is reported that the intraventricular administration of Casoxin C represented by r-Val-Leu-Ser-Arg suppresses the analgesic effect of opioids, and also shows an improvement effect on scopolamine-induced and amnesia due to cerebral ischemia. (Opioid Simposium 1
7, Supplement, 96-100).

In addition, the present inventors have disclosed a substance having a serum cholesterol-lowering effect as disclosed in JP-A-11-29289.
In 6, the X-Pro-Leu-Pro-Arg (X
Are Leu, Ile, Met, Phe, and Trp), and report that they have an effect of lowering blood cholesterol. Β-lactotensin (His-Il), which is a 4-residue ileal contraction peptide,
e-Arg-Leu) has been found to have a cholesterol-lowering effect upon oral administration. It is also known that a polymer peptide of unknown structure derived from soy protein has a cholesterol-lowering effect by adsorbing bile acid and inhibiting its reabsorption.

[0005]

In spite of these findings, the anti-amnesic effect can be more increased, considering that there are great differences between individuals in the pathology and manifestation of amnesia and hypercholesterolemia associated with aging. It is significant to obtain a substance having a cholesterol lowering effect. Also, considering that substances that are effective for senile diseases need to be taken for a long time,
It is desirable that oral administration is possible, and it is an object of the present invention to provide a functional peptide having such properties. In addition, a functional peptide that is effective for amnesia and lowers serum cholesterol has not been reported so far. It is also an object of the present invention to provide a peptide having both an anti-amnesic effect and a serum cholesterol-lowering effect in order to cope with an aging society.

[0006]

Means for Solving the Problems Accordingly, the present inventors have proposed p
enterostatin (Val-Pro-Asp-Pro), which is an endogenous peptide derived from E. rocolipase
-Arg). The peptide is known to exhibit an antifeeding action and an insulin secretion inhibitory action, and the present inventors have found that the peptide has a serum cholesterol lowering action as a new physiological function of enterostatin. He reported at the 1999 Agricultural Chemical Society meeting. Then, it was examined whether the fragment peptide of enterostatin also has a serum cholesterol lowering effect, and it was found that Pro-Asp-P
It was found that ro-Arg and Asp-Pro-Arg have a serum cholesterol lowering effect. The shorter the functional peptide, the less likely it is to be degraded in vivo and the easier it is to synthesize. Therefore, finding a shorter functional peptide fragment is significant.

[0007] Further, the findings that enterostatin inhibits the feeding-promoting action of β-casomorphine, which is a μ-opioid, suggesting that enterostatin has an action to antagonize the action of opioid receptors. Was done. Since opioids have a learning inhibitory effect, the effects of enterostatin on learning were examined from the viewpoint of anti-opioid effects. As a result, it was found that enterostatin was effective for the purpose of improving amnesia.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is an agent for preventing or improving amnesia, which comprises enterostatin or a fragment peptide thereof as an active ingredient. That is, Val-Pro-Asp-Pro-Arg,
Pro-Asp-Pro-Arg, Asp-Pro-A
An agent for preventing or improving amnesia, comprising a peptide represented by rg or cyclo (Asp-Pro) as an active ingredient.

[0009] Among the above peptides, Pro-Asp-
Pro-Arg and Asp-Pro-Arg have a serum cholesterol lowering effect. Therefore, the present invention
-Asp-Pro-Arg or Asp-Pro-Arg
It is a serum cholesterol lowering agent characterized by containing the peptide represented by these as an active ingredient. In the above, Val indicates valine, Pro indicates proline, Asp indicates aspartic acid, and Arg indicates arginine. All such amino acids are in the L-form.

[0010] 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.

In this reaction step, functional groups which should not take part 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 the 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 anti-alopecia agent of the present invention can be administered orally or parenterally. The dose of the peptide may vary depending on the type of compound, administration method, patient condition, age, etc.
Usually 0.1mg / kg to 1000mg / k per day
g, preferably 1 mg / kg to 100 mg / kg. 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, injections and the like.
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 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, the 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, 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.

In order to produce a rectal preparation, the active ingredient is dissolved together with a suppository base material such as cocoa butter, tri-, di- and monoglycerides of fatty acids, polyethylene glycol and the like, 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, etc., and if necessary, humidified and kneaded to form 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.

[0022]

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 put in a reaction vessel, and 1 mmol of Fmoc-Pr
o and 1 mmol of HBTU as an activator in 10 m
1 of dimethylformamide containing 0.4 M of N-methylmorpholine was added to the reaction vessel, and the mixture was stirred and reacted at room temperature for 20 minutes.

The Fmoc group was removed from the C-terminal of the obtained resin in the same manner as in the above-described method of coupling Fmoc-Pro in 20 ml of dimethylformamide containing 20% by weight of piperidine to remove the Fmoc group. Let them couple, Val-Pro-Asp (oBz
l) -Pro-Arg (Pmc) -resin was obtained. The resin was added to 10 ml of deprotection solution (82% by volume trifluoroacetic acid,
5% by volume thioanisole, 3% by volume ethanediol, 2
Volume% ethyl methyl sulfide, 3 volume% phenol,
The peptide was released from the resin by stirring at room temperature for 4 hours in 5% by volume water).

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
15C ₁₈ -AR, 20 × 250 mm), developed and purified with a linear concentration gradient of acetonitrile containing 0.1% by weight of trifluoroacetic acid by reverse phase chromatography.
Val-Pro-Asp-Pro-Arg was obtained. 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 addition, Pro-Asp-Pro-
Arg and Asp-Pro-Arg were also synthesized in the same manner. cyclo (Asp-Pro)
The synthesis of is shown below.

10 mmol of Boc-Asp (OBz
l) and Pro-OMe were dissolved in dichloromethane,
mmol of dicyclohexylcarbodiimide and hydroxybenzotriazole were added and reacted for 2 hours.
oc-Asp (OBzl) -Pro-OMe was obtained. This is washed with Asp (OBzl) -Pro in trifluoroacetic acid.
Cyclo (Asp (OB) was converted to OMe and cyclized in 2-butanol containing 1 M acetic acid for 3 hours.
zl) -Pro) was obtained. This was reduced with hydrogen in the presence of platinum black to obtain cyclo (Asp-Pro).

(Anti-amnestic action) The effect of the peptide on the learning ability was examined by a passive avoidance experiment using a step-through device. ddy mouse (male, weight 24 ± 2g)
When the mouse is placed in the light room of the device divided into two rooms, the mouse enters the dark room because it prefers dark places. When entering the dark room, electric shock (28-29V, 5 sec durat)
Ion) to educate that the darkroom is dangerous. 24
Time Retention of the memory was assessed by measuring the time the mice were put back into the same device and remained dark and light. Scopolamine amnesia was induced by intraperitoneally administering 0.1 mg / kg of scopolamine 30 minutes before the training walk. 100, 50 or 25 nmol /
Administering the peptide intracerebrally or orally at the dose of the mouse,
After 24 hours, a test trial was performed.

The effect of enterostatin intracerebral administration (100 nmol / mouse) on scopolamine-induced amnesia was examined. Time (step
FIG. 1 shows the results of examining the anti-amnestic effect of enterostatin, using -through latency: sec) as an index. Although the mouse has the property of moving to a dark room, it stays in the bright room if it remembers that the dark room is dangerous by learning, so it will be step-through
cy becomes longer. FIG. 1 shows that the step-through latency was recovered by administration of enterostatin, indicating that amnesia by scopolamine was improved as well as the control. In FIG. 1, n = 8, and Man and Whitney (Mann)
and Whitney), a difference was found between the control and ACSF (artificial cerebrospinal fluid) at a risk of 5%, and no difference was found between the control and the enterostatin-administered group.

Furthermore, enterostatin (Val-Pro)
-Asp-Pro-Arg), Pro-Asp-Pro
-Arg, Asp-Pro-Arg and cyclo (A
For sp-Pro), the anti-amnesic effect of oral administration was examined. As a result, they were effective against scopolamine-induced amnesia. Table 1 shows the minimum dose at which the above peptide exerts an anti-amnesic effect by oral administration. That is, n
= 8 with step-through with 5% risk factor
The minimum dose of peptide at which latency is restored is shown in Table 1.

[0029]

[Table 1]

It is known that the μ-opioid peptide has a learning inhibitory effect together with an anti-analgesic effect. Given that, the anti-amnesic effect of enterostatin may be provided by antagonizing the μ-opioid peptide. The present inventors have found that the above enterostatin and its derivative peptide antagonize the anti-analgesic action of morphine. The peptide of the present invention did not show an inhibitory effect on the analgesic effect of κ-opioid or δ-opioid. The above findings indicate that the anti-amnesic effect of the peptide of the present invention may be mediated by the μ-opioid receptor.

(Serum cholesterol-lowering effect) After pre-breeding ddy mice (male, body weight 20 g), they were fed a high cholesterol and high cholic acid diet, and were fed on days 0, 1, and 2.
On the day, the sample was orally administered, and on the third day, heart blood was collected.
After incubation at 37 ° C., serum was collected by centrifugation,
The total cholesterol concentration was measured by the enzyme method. Further, LDL and VLDL were precipitated using heparin to separate HDL, and HDL cholesterol was quantified. Heparin-precipitating lipoprotein (HPL) was determined by subtracting HDL cholesterol from total cholesterol. 0.6 for high cholesterol and high cholic acid diet
% Cholesterol and 0.2% cholic acid. The composition of the diet is shown in Table 2. LDL and VLD
It is said that the value of HPL including L is particularly closely related to the occurrence of ischemic heart disease.

[0032]

[Table 2]

The serum total cholesterol level and the value of heparin-precipitating lipoprotein were compared with PDPR (Pro-Asp).
FIG. 2 shows the effect of DPR (A-Pro-Arg).
The effect of sp-Pro-Arg) is shown in FIG. 3 (n = 8). As a result, both peptides had 100
At the administration of mg / kg, the total cholesterol value and the heparin-precipitating lipoprotein value were significantly different from the control at a risk rate of 5%. That is, the serum total cholesterol value is 20-30%, and the heparin-precipitating lipoprotein value is 30%.
A reduction of 低下 40% was observed, indicating that these peptides are effective orally in improving serum lipids. Since a functional peptide is preferably shorter in the sense of stability in vivo and ease of synthesis, it is of great significance that the enterostatin fragment is orally effective. Since the spectrum of the peptide having an anti-amnesic effect is different from that of the peptide having a serum cholesterol-lowering effect, it is considered that the anti-amnesic effect and the serum cholesterol-lowering effect are mediated by different mechanisms.

[0034]

EFFECT OF THE INVENTION Enterostatin (Val-Pro-A)
sp-Pro-Arg) and its fragment peptides (Pro-Asp-Pro-Arg, Asp-Pro-
Arg, cyclo (Asp-Pro)) has an anti-amnestic effect, and a functional peptide useful as a preventive or ameliorative agent for amnesia has been provided by the present invention. Furthermore, a fragment peptide of enterostatin (Pro-A
(Sp-Pro-Arg, Asp-Pro-Arg) have serum cholesterol-lowering agents, and functional peptides useful as serum cholesterol-lowering agents were also provided by the present invention.

[Brief description of the drawings]

FIG. 1 is a diagram showing the effect of enterostatin on scopolamine amnesia.

FIG. 2 is a graph showing the effect of PDPR on lowering serum cholesterol levels.

FIG. 3 is a graph showing the effect of DPR on lowering serum cholesterol levels.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 5/12 C07K 7/06 ZNA 7/06 ZNA A61K 37/02

Claims (6)

[Claims] 1. Val-Pro-Asp-Pro-Ar
An agent for preventing or improving amnesia, comprising a peptide represented by g as an active ingredient. 2. A preventive or ameliorative agent for amnesia comprising a peptide represented by Pro-Asp-Pro-Arg as an active ingredient. 3. An agent for preventing or improving amnesia, comprising a peptide represented by Asp-Pro-Arg as an active ingredient. 4. A preventive or ameliorative agent for amnesia comprising a peptide represented by cyclo (Asp-Pro) as an active ingredient. 5. A serum cholesterol lowering agent comprising a peptide represented by Pro-Asp-Pro-Arg as an active ingredient. 6. A serum cholesterol lowering agent comprising a peptide represented by Asp-Pro-Arg as an active ingredient.