JP2001226284A - Neurite inducer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provided a neurite inducer capable of safely using in clinical point of view without accompanying side effects such as bronchoconstrictive action or the like. SOLUTION: This neurite inducer includes a peptide or its pharmaceutically permissible salt as the active ingredient induced from PACAP(pituitary adenylate cyclase activating peptide) or VIP(vasoactive intestinal peptide). The pharmaceutical preparation is effective for improvement of Alzheimer's dementia, Parkinson's disease or the like and effective as a defense to medicine such as antitumor agent or the like imparting hazards to nervous system due to the neurite inducing effect.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a neurite-inducing agent useful for amelioration of Alzheimer's dementia, Parkinson's disease, etc., more specifically, VIP (vasoactive intestinal peptide) or PACAP (pituitary adenylate cyclase activity). And a pharmaceutically acceptable salt thereof as an active ingredient.

[0002]

2. Description of the Related Art VIP (vasoactive intestinal peptide; Voso)
Active Intestinal Peptide) is called a brain-intestinal peptide, and is a kind of a physiologically active peptide having blood flow promoting and blood pressure lowering effects. This VIP has been extracted from the pig intestinal tract and consists of 28 amino acid residues [SISaid, V. Mut.
t, Science., 169, 1217 (1970)]. Meanwhile, PACAP
(Pituitary adenylate cyclase activating peptide; Pitu
itary Adenylate Cyclase Activating Polypeptide)
Is a peptide consisting of 38 amino acid residues that was isolated and determined in structure from a bioassay system that activates adenylate cyclase in pituitary culture cells from the hypothalamus of sheep, and PACAP38 and PACAP27 There are two types [A. Miyata, A. Arimura et al., Biochem. Bio
phys. Res. Commun., 164, 567 (1989)]. This PACA
The 27 amino acid sequence from the N-terminal side of P has a structure very similar to VIP. Also, VIP and PACAP
Is similar to secretin, glucagon, etc., and is considered to be a peptide belonging to the glucagon-secretin superfamily.

[0003] When the activating action of pituitary adenylate cyclase in PACAP38 and PACAP27 is compared based on the accumulation of cAMP in the pituitary gland, no difference is observed between the two. However, PACAP requires only about 0.1 nM to activate adenylate cyclase, while VIP is 1 μM, and the concentration of PACAP is 1000 μM.
~ 10000 times as much [D. Spengler et al., N
ature, 365, 170-175 (1993)]. Thus, VIP and PACAP have a large difference in central activity,
There are few reports of differences in peripheral activity. PACAP receptors are mainly centrally located, VIP
Receptors mainly exist in the periphery, but the above difference is considered to be caused by different ligands recognized by these receptors. That is, PACAP receptor is used as a specific ligand for PACAP (PACA).
While only P38 and PACAP27) recognize, the VIP receptor recognizes both PACAP and VIP similarly. The reason why PACAP and VIP can bind to the same receptor is thought to be that both have a recognition site at their N-terminal site and that the amino acid sequences at these sites are similar as described above. [S. Onoue e
t al., Biomedical Research, 20 (4) 219-23 (1999)]
. Therefore, the present inventors have developed PACA based on such knowledge.
Synthesizing a VIP derivative exhibiting a P-like action and a PACAP derivative exhibiting a VIP-like action, and examining their physiological actions, it was found that these peptide derivatives have a strong bronchodilator action in common. Confirmed (Japanese Unexamined Patent Publication
-333276, JP-A-11-100399).

[0004] To date, specific binding sites for PACAP have been present in astrocytes (astroglia).
It has been reported that the action of ACAP promotes cAMP production [I. Tatsuno et al., B
iochem. Biophys. Res.Commun., 168, 1027-1033 (199
0)]. Astrocytes form scars in the brain, participate in the transport of substances between blood vessels and nerve cells (neurons), insulate synapses, take up released transmitters, and regulate the development and survival of neurons. Produces many neurotrophic factors. On the other hand, in Alzheimer-type dementia, neurofibrillary tangles occur in nerve cells, and a type of protein called amyloid is deposited, and degenerate nerve cells and senile plaques with astroglia and microglia accumulate around the nerve. It is a disease characterized by being created in the space between cells and nerve cells. Therefore, in view of the relationship between the above-mentioned astrocytes, the function of which is activated by the action of PACAP, PACAP is useful in the treatment of Alzheimer's dementia, which is an obstacle to the formation of neurons in the brain and the metabolism of nerve cells. It is considered useful for improvement. In fact, the effect of PACAP27, PAPAP38 and its derivatives on improving Alzheimer's disease through the action of secretogranin II production has been recognized (JP-A-6-2280).
02).

On the other hand, noradrenaline and further epinephrine are synthesized from tyrosine via dopa and dopamine from medulla chromaffin cells of the adrenal medulla. Noradrenaline and adrenaline (collectively catecholamines) are stored in granules of chromaffin cells,
It is secreted into the blood at the time of stress or struggle and reaches the target tissue to act. Phenochromcytoma is known as an abnormal catecholamine secretion, and is a disease in which catecholamine production is increased by tumors of chromaffin cells in the adrenal medulla or paraganglia. Cells derived from the pheochromocytoma of the rat adrenal medulla are called PC-12. These cells contain noradrenaline, adrenaline, and the neuropeptide enkephalin in chromaffin granules equivalent to synaptic vesicles, and acetylcholine (Ach) released from splanchnic nerve endings
Secretes these hormones and peptides in response to stimuli. In addition, embryologically, it is homologous to the postganglionic nerve cells of the sympathetic nervous system, and the adrenal medulla is composed of a nearly equal group of paraneuronal cells. In PC-12, the basic pathway of a series of reactions including ion influx after stimulus reception, further secretion of catecholamines, and short-term recovery to the prestimulatory state has been clarified. Occurs. Therefore, PC-12 has been established as a model system in brain and nervous system research.
A number of neurite-inducing substances have been reported by searching with C-12, including PACAP [T. Watanabe et a
l., Biochem. Biophys. Res.Com., 182, 403-411 (199
2)].

[0006] As one of the treatments for Parkinson's disease, attempts have been made to improve clinical symptoms by administering L-DOPA to increase dopamine in the brain, but the secretion of catecholamines as described above has been attempted. Neurite induction associated with promotion is also thought to be effective in treating such diseases. In addition, some antitumor agents, such as vin alkaloid antitumor agents such as vincristine, platinum complex antitumor agents such as cisplatin, adriamycin antitumor agents, taxol antitumor agents, etc., impair neurite formation Is known to cause. This develops into peripheral neuropathy, and various symptoms may appear, such as mild swelling of the limbs to severe limb paralysis requiring care in daily life. It has been pointed out that NGF (Nerve Growth Factor), which induces neurites, is effective in improving peripheral neuropathy caused by administration of the above antitumor agent [SCApfel et a
l., Ann. Neurol., 29, 87-90 (1991)], and a similar effect of improving peripheral neuropathy has been observed with PACAP (Japanese Patent Application Laid-Open No. Hei 8-3056).

As described above, since PACAP has a neurite-inducing effect, it is effective in improving Alzheimer's dementia, Parkinson's disease, etc., and in protecting against drugs such as antitumor agents that damage the nervous system. It is possible,
PACAP has a transient bronchoconstrictive effect, which is a fatal side effect for patients with bronchial asthma and similar allergies.

[0008]

Accordingly, an object of the present invention is to provide a neurite-inducing agent which has no side effects such as bronchoconstriction and can be used safely clinically.

[0009]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, a series of VIP, P
The inventors have found that ACAP peptide derivatives exert a neurite-inducing action without causing side effects unlike PACAP, and have completed the present invention. That is, the present invention is the following inventions (1) to (3). (1) The following formula (I):

[0010]

Embedded image H-His-Ser-Asp-AB-Phe-Thr-Asp-C-Tyr-D-Arg-E-Arg-F-Gln-G-Ala-Val-I- (I) J-Tyr -Leu-KLM-Leu-N

Wherein A is Ala or Gly; B is Il
e or Val; C is Asn or Ser; D is Thr or Ser; E is Leu or Tyr; F, I, J is
Lys or Arg; G is Met, Leu or nLeu; K is
Asn or Ala; L is, Ser or Ala; M is Ile or Val; N represents a -NH ₂ or Asn-NH _2. However,
At the same time, A is Ala, B is Val, C is Asn, D is Thr, and E is L
eu, K is Asn, L is Ser, M is Ile and N is Asn-NH ₂
Never be. At the same time, A is Gly, B is Ile, C
Is Ser, D is Ser, E is Tyr, K is Ala, L is Ala, M
There never Val and N is -NH _2. ) Or a pharmaceutically acceptable salt thereof as an active ingredient.

(2) The following formula (II):

[0013]

Embedded image H-His-Ser-Asp-AB-Phe-Thr-Asp-C-Tyr-D-Arg-E-Arg-F-Gln-G-Ala-Val-I- (II) J-Tyr -Leu-KLM-Leu-P-Gly-QR

Wherein A is Ala or Gly; B is Il
e or Val; C is Asn or Ser; D is Thr or Ser; E is Leu or Tyr; F, I, J is
Lys or Arg; G is Met, Leu or nLeu; K is
Asn or Ala; L is Ser or Ala; M is Ile or Val; P is Asn or a chemical bond; Q is Lys, Ar
g, Lys-Arg, Arg- Arg or a chemical bond; R represents an -OH or -NH _2. ) Or a pharmaceutically acceptable salt thereof as an active ingredient.

(3) The following formula (III):

[0016]

Embedded image H-His-Ser-Asp-AB-Phe-Thr-Asp-C-Tyr-D-Arg-E-Arg-F-Gln-G-Ala-Val-I-J-Tyr-Leu- KLM-Leu-N-Gly-OP- (III) Tyr-Q-Gln-R-Val-S-Asn-TU

(Where A is Ala or Gly; B is Il
e or Val; C is Asn or Ser; D is Thr or Ser; E is Leu or Tyr; I is Lys or Arg or Gln; F, J, O, P, Q, R, S, T is Lys or Arg; at least one of F, I and J is Arg; G is
Met, Leu or nLeu; K is Asn or Ala; L is
Ser or Ala; M is Ile or Val; N is a chemical bond or Asn; U represents an -OH or -NH _2. However, at the same time, A is Gly, B is Ile, C is Ser, D is Ser, E is Ty
r, K is Ala, L is Ala, M is Val, N is a chemical bond, O
Is Lys, P is Arg, Q is Lys, R is Arg, S is Lys, T
There Lys, U never becomes -NH _2. ) Or a pharmaceutically acceptable salt thereof as an active ingredient. Hereinafter, the present invention will be described specifically.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide derivative which is the active ingredient of the neurite-inducing agent of the present invention is specifically a peptide having the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1. Of the peptide consisting of
A peptide consisting of an amino acid sequence having Asn or the amino acid sequence of any of SEQ ID NOs: 2 to 11 added to its terminal.

[0019]

Embedded image His-Ser-Asp-Xaa-Xaa-Phe-Thr-Asp-Xaa-Tyr-Xaa-Arg-Xa
a-Arg-Xaa-Gln- Xaa-Ala-Val-Xaa-Xaa-Tyr-Leu-X
aa-Xaa-Xaa-Leu (SEQ ID NO: 1) Gly-Lys (SEQ ID NO: 2) Gly-Arg (SEQ ID NO: 3) Gly-Lys-Arg (SEQ ID NO: 4) Gly-Arg-Arg (SEQ ID NO: 5) Asn- Gly-Lys (SEQ ID NO: 6) Asn-Gly-Arg (SEQ ID NO: 7) Asn-Gly-Lys-Arg (SEQ ID NO: 8) Asn-Gly-Arg-Arg (SEQ ID NO: 9) Gly-Xaa-Xaa-Tyr- Xaa-Gln-Xaa-Val-Xaa-Asn-Xaa (SEQ ID NO: 10) Asn-Gly-Xaa-Xaa-Tyr-Xaa-Gln-Xaa-Val-Xaa-Asn-Xaa
(SEQ ID NO: 11)

However, of the above peptides, peptides having the following amino acid sequence are excluded.

[0021]

Embedded image His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-
Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Ly
s (SEQ ID NO: 12) His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-
Val-Leu (SEQ ID NO: 13) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn (SEQ ID NO: 14)

Hereinafter, the present invention will be described in detail.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide used in the neurite-inducing agent of the present invention can be synthesized according to a known conventional method for peptide synthesis. For example, "The Peptides"
Volume 1 (1966) (Schreder and Luhke, Academic Pre
ss, New York, USA] or “peptide synthesis”
[Izumiya et al., Maruzen Co., Ltd. (1975)], specifically, azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method (P-nitro Phenyl ester method, N-hydroxysuccinimide ester method, cyanomethyl ester method, etc.), method using Woodward reagent K, carboimidazole method, redox method, DCC-additive (HONB, HOBt, HO)
It can be synthesized by various methods such as the Su) method. These methods can be applied to both solid phase synthesis and liquid phase synthesis.

In the present invention, peptide synthesis is carried out according to the general method for synthesizing a polypeptide as described above, for example, by the so-called stepwise method in which amino acids are sequentially condensed one by one with terminal amino acids, or by dividing into several fragments. It is performed by a coupling method.

For example, the solid phase synthesis by the stepwise method is specifically performed by the method of Merrifield (RB) [Solid phase peptide synthesis, J. Amer. Chem. S
oc., 85, 2149-2159 (1963)]. First, a C-terminal amino acid (having an amino group protected) is bound to an insoluble resin via its carboxyl group, and then the amino-protecting group of the C-terminal amino acid is removed. Next, a reactive carboxyl group of the amino group-protected amino acid is sequentially bonded to the obtained free reactive amino group by a condensation reaction in accordance with the amino acid sequence of the target peptide. After synthesizing the entire sequence step by step in this way, the peptide is removed from the insoluble resin.

As the insoluble resin used in the above-mentioned solid phase synthesis, any one having a binding property to a reactive carboxyl group can be used, for example, benzhydrylamine resin (BHA resin), chloromethyl resin, oxymethyl resin. Resin, aminomethyl resin, methylbenzhydryl resin (MBHA resin), 4-aminomethylphenoxymethyl resin, 4-hydroxymethylphenoxymethyl resin, 4-
Oxymethylphenylacetamide methyl resin and the like can be mentioned. When a 9-fluorenylmethyloxycarbonyl group (Fmoc) is used as a protecting group for an α-amino group, 4-hydroxymethylphenoxymethyl resin or the like is used.
Those which can be removed from the resin by trifluoroacetic acid (TFA) are good, and when a t-butoxycarbonyl group (Boc) is used, 4-oxymethylphenylacetamidomethyl resin (PAM resin), etc. Those that can be detached from the resin are preferred. The peptide concentration is 0.5mmo per 1g of resin
It is preferably set to le or less.

In the above-mentioned method, it is necessary to bond a protecting group to an amino group involved in the peptide bond of an amino acid and to remove the protecting group, and to activate a carboxyl group involved in the peptide bond of the amino acid.

Examples of the amino-protecting group include benzyloxycarbonyl (Z), t-butoxycarbonyl (Boc), t-aminooxycarbonyl (Aoc), isobonyloxycarbonyl, p-methoxybenzyloxycarbonyl, Chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, o-nitrophenylsulfenyl,
And groups such as diphenylphosphinothioyl.

Among the amino acids, those having a functional group in the side chain, such as His, Tyr, Thr, Lys, Asp, Arg and Se
r preferably protects the functional group of its side chain.
The protection of the functional group is performed by bonding a usual protecting group as described below by a generally used method, and after completion of the reaction, the protecting group is eliminated. Examples of the protecting group for the imino group of His include benzyloxymethyl (Bom) and p-
Examples include toluenesulfonyl (Tos), benzyl (Bzl), benzyloxycarbonyl (Z), and a trityl group.

The hydroxyl groups of Ser and Thr can be protected, for example, by esterification or etherification, but this protection is not essential. Groups suitable for esterification include lower alkanoyl groups such as acetyl, aroyl groups such as benzoyl, and groups derived from carbonic acid such as benzoyloxycarbonyl and ethyloxycarbonyl. Groups suitable for etherification include benzyl (Bzl), tetrahydropyranyl, tert-butyl and the like.

Examples of the protecting group for the hydroxyl group of Tyr include benzyl (Bzl) and bromobenzyloxycarbonyl (Br-
Z), dichlorobenzyl (Cl ₂ -Bzl), benzyloxycarbonyl (Z), acetyl, p-toluenesulfonyl (To
s) groups and the like. The protecting group of the amino groups of Lys, for example, benzyloxycarbonyl (Z), chlorobenzyloxycarbonyl (Cl-Z), dichlorobenzyl (Cl ₂ -Bzl), t- butoxycarbonyl group (Boc), p-toluenesulfonyl (Tos) group and the like.

Examples of the protecting group for the guanidino group of Arg include p-toluenesulfonyl (Tos), nitro, benzyloxycarbonyl (Z), t-amyloxycarbonyl (Aoc) and the like. The protection of the carboxyl group of Asp is performed by, for example, esterification with benzyl alcohol, methanol, ethanol, tert-butanol, cyclohexyl (cHex) or the like.

Other protecting groups for amino acids, such as protecting group for the indolyl group of Trp, include, for example, formyl, carbobenzoxyl, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, 2,2,2-trichloroethyl Oxycarbonyl and the like can be mentioned, but this protection is not essential. Met
As a protecting group for the thiomethyl group of the above, there is a method in which methyl sulfoxide is previously formed and then reduced, but this protection is not essential.

On the other hand, the activation of the carboxyl group can be performed by a conventionally known method, and the reagents and the like to be used can be appropriately selected from known ones. For example, activation of a carboxy group can be achieved by reacting the carboxyl group with various reagents to form the corresponding acid chloride, acid anhydride or mixed acid anhydride, azide, active ester (pentachlorophenol,
p-nitrophenol, N-hydroxysuccinimide,
N-hydroxybenztriazole, N-hydroxy-
(An ester with 5-norbornene-2,3-dicarboximide) or the like.

The solvent used for the condensation reaction (peptide bond formation reaction) between the reactive amino group and the reactive carboxyl group on the solid phase may be any solvent that can be used for peptide bond formation. For example, anhydrous or water-containing dimethylformamide (DMF), dimethylsulfoxide (DMSO), pyridine, chloroform, dioxane, dichloromethane (DCM), tetrahydrofuran (THM
F), ethyl acetate, N-methylpyrrolidone, hexamethylphosphoric triamide (HMPA) and the like can be used alone or as a mixed solvent of two or more.

In the above condensation reaction, a condensing agent such as a carbodiimide reagent such as dicyclohexylcarboximide (DCC) or carbodiimidazole, tetraethyl pyrophosphate, benzotriazole-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide ( (Bop reagent) or the like.

The synthesized peptide can be desalted and purified according to a conventional method. For example, ion exchange chromatography such as DEAE-cellulose, partition chromatography such as Sephadex LH-20, Sephadex G-25, normal phase chromatography such as silica gel,
Examples include reverse phase chromatography such as ODS-silica gel, high performance liquid chromatography, and the like.

The peptide purified as described above can be converted into a pharmaceutically acceptable salt, for example, acetate, hydrochloride, phosphate and the like, if desired, using various acids. The above-mentioned peptide and salts thereof are prepared by using a pharmaceutically acceptable solvent, excipient, carrier, auxiliary agent and the like, and in accordance with a conventional method for the production of a liquid, injection, tablet, powder, granule, suppository, enteric solvent. And pharmaceutical preparations such as capsules.

The above peptides and salts thereof are useful as neurite-inducing agents because they have a neurite-inducing effect.
The preparation is effective for prevention and treatment of various diseases, specifically, Alzheimer's disease, Parkinson's disease, neuronal cell death, neuroblastoma, amnesia, and protection against drugs that damage the nervous system. .

The preparation can be safely administered parenterally or orally to mammals such as humans, mice, rats, rabbits, dogs and cats. The dose of the preparation can be appropriately changed depending on the dosage form, administration route, symptoms, etc., for example, when administered to mammals including humans, the amount of peptide per person per day is about 1 pg to 1 mg / kg.
Applying a degree is exemplified.

[0041]

EXAMPLES The present invention will be described more specifically with reference to examples and working examples, but the present invention is not limited to these examples.

Reference Example 1 (Synthesis of Peptide) PACAP38, PACAP27, VIP having the following amino acid sequences
And peptide derivatives (peptides 1 to 14) derived therefrom were synthesized according to the usual peptide solid phase synthesis method. Incidentally, N-terminus of each peptide is -H, a C-terminal is -NH _2.

(A) PACAP38 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-
Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Ly
s (SEQ ID NO: 12) (b) PACAP27 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-
Val-Leu (SEQ ID NO: 13) (c) VIP His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn (SEQ ID NO: 14) (d) Peptide 1 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-
Val-Leu (SEQ ID NO: 15) (e) Peptide 2 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-
Val-Leu (SEQ ID NO: 16) (f) Peptide 3 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-
Val-Leu (SEQ ID NO: 17) (g) Peptide 4 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Ser-Tyr-Thr-Arg-Le
u-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn (SEQ ID NO: 18) (h) Peptide 5 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn (SEQ ID NO: 19) (i) Peptide 6 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Thr-Arg-Le
u-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn (SEQ ID NO: 20) (j) Peptide 7 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn (SEQ ID NO: 21) (k) Peptide 8 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn-Gly-Lys-Arg (SEQ ID NO: 22) (l) Peptide 9 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Ala-Ala-
Val-Leu (SEQ ID NO: 23) (m) Peptide 10 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Ala-Ala-
Val-Leu-Gly-Lys-Arg (SEQ ID NO: 24) (n) Peptide 11 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Ala-Ala-
Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Ly
s (SEQ ID NO: 25) (o) Peptide 12 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Ala-Ala-
Val-Leu-Gly-Arg-Arg-Tyr-Arg-Gln-Arg-Val-Arg-Asn-Ar
g (SEQ ID NO: 26) (p) Peptide 13 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-As
n-Lys (SEQ ID NO: 27) (q) peptide 14 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Le
u-Arg-Arg-Gln-Leu-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-
Ile-Leu-Asn-Gly-Arg-Arg-Tyr-Arg-Gln-Arg-Val-Arg-As
n-Arg (SEQ ID NO: 28) (r) Peptide 15 His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-Met-Ala-Val-Gln-Lys-Tyr-Leu-Ala-Ala-
Val-Leu-Gly-Arg-Arg-Tyr-Arg-Gln-Arg-Val-Arg-Asn-Ly
s (SEQ ID NO: 29)

Example 1 Neurite Inducing Action Test The neurite inducing action of the peptide synthesized in Reference Example 1 was tested according to the following method. 5% PC-12 cells
Dulbecco's modi containing horse serum and 5% newborn calf serum
fiedminimum essential 5% in the medium (DMEM) CO _2/95
The culture was maintained at 37 ° C in an environment of% air. After detaching the cells from the culture flask with trypsin, the cells were counted with a hemocytometer and adjusted to 5.0 × 10 ⁴ cells / ml. Add 4 ml of the above cell-containing liquid medium to a 6 mm collagen type IV biocoat dish for 24 hours.
The cells were cultured at 37 ° C. in an environment of 5% CO ₂ /95% air.
After 24 hours, the medium was changed, and each peptide (100 nM)
Was added. This was continued for three days, four sections were selected from two dishes, photographed, and then converted to TIFF file format using a scanner. Next, the area of the neurite portion was measured for this file using image processing software. The peptide-free sample was used as a control,
The data for each sample is shown as a ratio (%) to the control. Table 1 shows the results.

[0045]

[Table 1]

From these results, it is clear that a series of peptide derivatives have a neurite-inducing effect, and it is clear that VIP derivatives, which should have low activity, also have high activity by partial structural conversion. became.

Example 2 (Bronchial Dilation Test) The peptides 2 and 5 synthesized in Reference Example 1 were tested for the presence or absence of a temporary contraction effect during bronchial smooth muscle dilation according to the following method. Hartley male guinea pig (350
450450 g), 6-7 weeks old (Japan SLC) were beaten and stunned, exsanguinated via the femoral artery, and thoracotomy was performed to remove the trachea.
Adipose tissue adhering to the trachea was removed as much as possible, and the cartilage portion on the opposite side of the esophagus was cut vertically, and then cut along the cartilage to a width including two cartilage pieces to obtain a section. One section was selected for each of the upper, middle and lower respiratory tracts, and each piece was tied with a silk thread at the cartilage portion to obtain a chain-shaped section. Specimens were placed in a Magnus bath (volume 5 ml, temperature 37 ° C, load 1.5 g, 95% O ₂ + 5
(Under aeration of% CO ₂ ). Krebs solution was used as a physiological solution. The reaction is isometric transducer (TB-612 T,
TB-611 T: Nihon Kohden using amplifier (RMP-6018
M: Depicted on recorder (WT-685 G, Nihon Kohden) via Nihon Kohden. After the contraction by acetylcholine 10 ^-5 M was stabilized, each test peptide (3 x 10 ^-7 M) was administered to one sample at a concentration, and after measuring for 3 hours, isoproterenol (isoprenol)
(roterenol) 10 ^-6 M was administered, and the relaxation rate of each test substance was determined by taking the value as 100% relaxation. As a result, PACAP 27, P
ACAP 38 was found to have a temporary contractile action immediately after administration. However, neither the PACAP peptide derivative (peptide 2) nor the VIP peptide derivative (peptide 5) showed such an effect, and showed only a pure bronchodilator effect. This indicates that even in patients with bronchial asthma or allergies equivalent thereto, a series of peptide derivatives as active ingredients in the present invention does not exhibit bronchoconstrictive action which may be a side effect and can be used safely.

[0048]

According to the present invention, there is provided a neurite-inducing agent which has no side effects such as bronchoconstriction and can be used safely clinically. This preparation is effective for amelioration of Alzheimer's dementia, Parkinson's disease, etc., and protection against drugs such as antitumor agents that damage the nervous system by neurite-inducing action.

[0049]

[Sequence List] SEQUENCE LISTING <110> ITOHAM FOODS INC. <120> Agent for inducing neurite <130> P99-0701 <160> 29 <170> PatentIn Ver. 2.0 <210> 1 <211> 27 <212> PRT < 213> Artificial Sequnece <220> <223> Synthetic Peptide <220> <221> PEPTIDE <222> 4 <223> Xaa represents Ala or Gly <220> <221> PEPTIDE <222> 5 <223> Xaa represents Ile or Val <220> <221> PEPTIDE <222> 9 <223> Xaa represents Asn or Ser <220> <221> PEPTIDE <222> 11 <223> Xaa represents Thr or Ser <220> <221> PEPTIDE <222> 13 < 223> Xaa represents Leu or Tyr <220> <221> PEPTIDE <222> 15 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 17 <223> Xaa represents Met or Leu or nLeu <220> <221> PEPTIDE <222> 20 <223> Xaa represents Lys or Arg or Gln <220> <221> PEPTIDE <222> 21 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 24 <223 > Xaa represents Asn or Ala <220> <221> PEPTIDE <222> 25 <223> Xaa represents Ser or Ala <220> <221> PEPTIDE <222> 26 <223> Xaa represents Ile or Val <400> 1 His Ser Asp Xaa Xaa Phe T hr Asp Xaa Tyr Xaa Arg Xaa Arg Xaa Gln 1 5 10 15 Xaa Ala Val Xaa Xaa Tyr Leu Xaa Xaa Xaa Leu 20 25 <210> 2 <211> 2 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 2 Gly Lys 1 <210> 3 <211> 2 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 3 Gly Arg 1 <210> 4 <211> 3 < 212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 4 Gly Lys Arg 1 <210> 5 <211> 3 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide < 400> 5 Gly Arg Arg 1 <210> 6 <211> 3 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 6 Asn Gly Lys 1 <210> 7 <211> 3 <212 > PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 7 Asn Gly Arg 1 <210> 8 <211> 4 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400 > 8 Asn Gly Lys Arg 1 <210> 9 <211> 4 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 9 Asn Gly Arg Arg 1 <210> 10 <211> 11 < 212> PRT <213> Artificial Sequnece <220> <223> Sy nthetic Peptide <220> <221> PEPTIDE <222> 2 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 3 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 5 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 7 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 9 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 11 <223> Xaa represents Lys or Arg <400> 10 Gly Xaa Xaa Tyr Xaa Gln Xaa Val Xaa Asn Xaa 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <220> <221> PEPTIDE <222> 3 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 4 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 6 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 8 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 10 <223> Xaa represents Lys or Arg <220> <221> PEPTIDE <222> 12 <223> Xaa represents Lys or Arg <400> 11 Asn Gly Xaa Xaa Tyr Xaa Gln Xaa Val Xaa Asn Xaa 1 5 10 <210> 12 <211> 38 <212> PRT <213> Artificial Sequne ce <220> <223> Synthetic Peptide <400> 12 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys 20 25 30 Gln Arg Val Lys Asn Lys 35 <210> 13 <211> 27 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 13 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 14 <211> 28 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 14 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 <210> 15 <211> 27 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 15 His Ser Asp Gly Ile Phe Thr Asp Asn Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 16 <211> 27 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 16 His Ser Asp Ala Val Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 17 <211> 27 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide < 400> 17 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu 20 25 <210> 18 <211> 28 <212> PRT <213 > Artificial Sequnece <220> <223> Synthetic Peptide <400> 18 His Ser Asp Ala Val Phe Thr Asp Ser Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 < 210> 19 <211> 28 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 19 His Ser Asp Gly Ile Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 <210> 20 <211> 28 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 20 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 <210> 21 <211> 28 <212> PRT <21 3> Artificial Sequnece <220> <223> Synthetic Peptide <400> 21 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Asn Ser Ile Leu Asn 20 25 <210> 22 <211> 31 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 22 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Asn Ser Ile Leu Asn Gly Lys Arg 20 25 30 <210> 23 <211> 27 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 23 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Ala Ala Val Leu 20 25 <210> 24 <211> 30 <212> PRT <213> Artificial Sequnece <220> < 223> Synthetic Peptide <400> 24 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Ala Ala Val Leu Gly Lys Arg 20 25 30 <210> 25 < 211> 38 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 25 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys 20 25 30 Gln Arg Val Lys Asn Lys 35 <210> 26 <211> 38 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 26 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Ala Ala Val Leu Gly Arg Arg Tyr Arg 20 25 30 Gln Arg Val Arg Asn Arg 35 <210> 27 <211> 39 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 27 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Asn Ser Ile Leu Asn Gly Lys Arg Tyr 20 25 30 Lys Gln Arg Val Lys Asn Lys 35 <210> 28 <211> 39 <212> PRT <213> Artificial Sequnece <220> <223> Synthetic Peptide <400> 28 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Arg Gln 1 5 10 15 Leu Ala Val Arg Arg Tyr Leu Asn Ser Ile Leu Asn Gly Arg Arg Tyr 20 25 30 Arg Gln Arg Val Arg Asn Arg 35 <210> 29 <211> 38 <212> PRT <213> Artificial Se qunece <220> <223> Synthetic Peptide <400> 29 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Gln Lys Tyr Leu Ala Ala Val Leu Gly Arg Arg Tyr Arg 20 25 30 Gln Arg Val Arg Asn Lys 35

[0050]

[Sequence listing free text] SEQ ID NO: 1: Xaa is Ala or
SEQ ID NO: 1: Xaa represents Ile or Val representing Gly (location: 4)
5) SEQ ID NO: 1: Xaa represents Asn or Ser (location:
9) SEQ ID NO: 1: Xaa represents Thr or Ser (location: 1)
1) SEQ ID NO: 1: Xaa represents Leu or Tyr (location: 1
3) SEQ ID NO: 1: Xaa represents Lys or Arg (location: 1)
5) SEQ ID NO: 1: Xaa represents Met or Leu or nLeu (location: 17) SEQ ID NO: 1: Xaa represents Lys, Arg, or Gln (location: 20) SEQ ID NO: 1: Xaa represents Lys or Arg Represent (location: 2
1) SEQ ID NO: 1: Xaa represents Asn or Ala (location: 2)
4) SEQ ID NO: 1: Xaa represents Ser or Ala (location: 2)
5) SEQ ID NO: 1: Xaa represents Ile or Val (location: 2)
6)

SEQ ID NO: 10: Xaa represents Lys or Arg (location: 2) SEQ ID NO: 10: Xaa represents Lys or Arg (location:
3) SEQ ID NO: 10: Xaa represents Lys or Arg (location:
5) SEQ ID NO: 10: Xaa represents Lys or Arg (location:
7) SEQ ID NO: 10: Xaa represents Lys or Arg (location:
9) SEQ ID NO: 10: Xaa represents Lys or Arg (location: 1)
1)

SEQ ID NO: 11: Xaa represents Lys or Arg (location: 3) SEQ ID NO: 11: Xaa represents Lys or Arg (location:
4) SEQ ID NO: 11: Xaa represents Lys or Arg (location:
6) SEQ ID NO: 11: Xaa represents Lys or Arg (location:
8) SEQ ID NO: 11: Xaa represents Lys or Arg (location: 1)
0) SEQ ID NO: 11: Xaa represents Lys or Arg (location: 1)
2)

[Brief description of the drawings]

FIG. 1 shows bronchial smooth muscle contraction by VIP, PACAP and their peptide derivatives.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C084 AA02 BA01 BA04 BA08 BA19 BA23 CA21 CA22 CA25 CA28 DB59 MA16 MA31 MA35 MA37 MA41 MA43 MA52 MA56 MA60 MA66 NA06 NA14 ZA011 ZA021 ZA151 ZA161 4H045 AA30 BA18 BA19 CA40 EA21 FA33

Claims (3)

[Claims] 1. The following formula (I): H-His-Ser-Asp-AB-Phe-Thr-Asp-C-Tyr-D-Arg-E-Arg-F-Gln-G-Ala-Val-I -(I) J-Tyr-Leu-KLM-Leu-N (where A is Ala or Gly; B is Ile or Val
; C is Asn or Ser; D is Thr or Ser; E
Is Leu or Tyr; F, I, J are Lys or
Arg; G is Met, Leu or nLeu; K is Asn or
Ala; L is Ser or Ala; M is Ile or Val;
N denotes the -NH ₂ or Asn-NH _2. However, at the same time A is A
la, B is Val, C is Asn, D is Thr, E is Leu, K is As
n, L is Ser, M is Ile, and N is not Asn-NH ₂ . At the same time, A is Gly, B is Ile, C is Ser, D
There Ser, E is Tyr, K is Ala, L is Ala, M never Val and N is -NH _2. ) Or a pharmaceutically acceptable salt thereof as an active ingredient. 2. The following formula (II): H-His-Ser-Asp-AB-Phe-Thr-Asp-C-Tyr-D-Arg-E-Arg-F-Gln-G-Ala-Val-I -(II) J-Tyr-Leu-KLM-Leu-P-Gly-QR (where A is Ala or Gly; B is Ile or Val
; C is Asn or Ser; D is Thr or Ser; E
Is Leu or Tyr; F, I, J are Lys or
Arg; G is Met, Leu or nLeu; K is Asn or
Ala; L is Ser or Ala; M is Ile or Val;
P is Asn or a chemical bond; Q is Lys, Arg, Lys-Arg,
Arg-Arg or a chemical bond; R represents an -OH or -NH _2. ) Or a pharmaceutically acceptable salt thereof as an active ingredient. 3. The following formula (III): H-His-Ser-Asp-AB-Phe-Thr-Asp-C-Tyr-D-Arg-E-Arg-F-Gln-G-Ala-Val-I -J-Tyr-Leu-KLM-Leu-N-Gly-OP- (III) Tyr-Q-Gln-R-Val-S-Asn-TU (where A is Ala or Gly; B is Ile Or Val
; C is Asn or Ser; D is Thr or Ser; E
Is Leu or Tyr; I is Lys or Arg or Gln;
F, J, O, P, Q, R, S, T are Lys or Arg respectively;
At least one of F, I and J is Arg; G is Met, Leu or nLeu; K is Asn or Ala; L is Ser or
Ala; M is Ile or Val; N is a chemical bond or A
sn; U represents an -OH or -NH _2. However, at the same time A is Gly,
B is Ile, C is Ser, D is Ser, E is Tyr, K is Ala,
L is Ala, M is Val, N is a chemical bond, O is Lys, P is
Arg, Q is Lys, R is Arg, S is Lys, T is Lys, U is-
Not to become a NH _2. ) Or a pharmaceutically acceptable salt thereof as an active ingredient.