JPH0616564A - Peptide for promoting production of nerve growth factor - Google Patents

Abstract

PURPOSE:To provide a drug for promoting production of a nerve growth factor, capable of mass-production at low cost. CONSTITUTION:The drug for promoting production of a nerve growth factor contains a peptide (The N-terminal amino group of the peptide represented by each sequence number may be modified with an acetyl group, a tert- butoxycarbonyl group or a benzyloxycarbonyl group and the C-terminal carboxyl group may be an amide group) expressed by sequence number 1 to 3 (formula I tok III) as the active component. The above-mentioned peptides can readily be obtained by chemical synthesis and mass-production of NGF is possible. Accordingly, this drug is hopeful as a medicine for improving a cerebral nerve disease such as dementia and a peripheral dysfunction due to peripheral lesion.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to nerve growth factor (hereinafter referred to as N
(Referred to as GF) Production promoter.

[0002]

NGF differentiates nerve cells in vitro, promotes neurite outgrowth, and maintains nerve cell survival. In animal experiments, administration of NGF into the brain has the effects of enhancing memory and learning ability and preventing death of neurons due to cerebral ischemia [J. Neurosci., 6, 21.
55 (1986), Brain Res., 293, 305 (1985), Science, 2
35, 214 (1986), Proc. Natl. Acad. Sci. USA., 83, 9
231 (1986), Annals of Neurology, 120, 275 (198
6)].

It has been reported that NGF is essential for survival and differentiation of these nerve cells [Pharmacia, 147 (1986),
Geriatric Psychiatry, 3, 751 (1986)], suggesting that NGF is a therapeutic drug for Alzheimer-type senile dementia in which most nerve cells (cholinergic neurons of the Meinert nucleus) that control memory and thought are killed and disappeared. Has been suggested as a possibility. In fact, according to a report by Lance-Olson et al. (1991 Alzheimer's Disease Treatment Symposium), NGF taken from a mouse was administered for a total of 6.6 mg for 3 months to the brain of a patient showing dementia symptoms due to Alzheimer's disease. Improvement was noted.

[0004] In the striatum of the brain of Huntington's chorea, the loss of GABAergic neurons and the loss of cholinergic neurons are remarkable, and NGF also acts on the endogenous cholinergic neurons of the striatum. It is known [Science, 234, 1341 (1986)] that NGF attracts attention as a therapeutic agent for cranial nerve diseases.

[0005] In peripheral nerves, NGF secretion of Schwann cells and fibroblast-like cells is enhanced with nerve damage [Proc. Natl. Acad. Sci. USA., 84, 8735 (1
987), J. Cell. Biol., 104, 1623 (1987), Nature, 3
30,658 (1987)], the relationship between peripheral nerve repair and regeneration and NGF has been studied in detail, and is expected as a therapeutic drug for peripheral nerve dysfunction such as spinal cord injury and peripheral nerve injury.

[0006]

A large amount of NGF is required for clinical use of this NGF.
Since the mass production technology means has not been established, NG
A substance that activates F-producing cells and promotes secretion of NGF is desired.

[0007]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that the peptide represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (represented by each SEQ ID NO: The N-terminal amino group of the prepared peptide may be modified with an acetyl group, a t-butoxycarbonyl group or a benzyloxycarbonyl group, and the C-terminal carboxyl group may be an amide group). The present invention has been completed and the present invention has been completed.

The peptide of the present invention can be easily obtained by applying a general peptide synthesis method. Peptide synthesis methods include activated ester method, mixed acid anhydride method, C-terminal activation method such as azide method, coupling method such as carbodiimide, N-carboxy anhydride (NCA) method, redox method,
Enzymatic method or solid-phase method developed by Merrifield [Angew. C
hem. Int. Ed. Engl., 24, 799 (1985)].

The peptide used in the present invention may be used in the form of a physiologically acceptable salt. The physiologically acceptable salts include alkali metal salts or alkaline earth metal salts, salts with amines and organic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, methanesulfonic acid, citric acid,
Salts such as tartaric acid, lactic acid, oleic acid, fumaric acid, malic acid, cinnamic acid, malonic acid, glutamic acid, aspartic acid, mucous acid, benzoic acid, gluconic acid, oxalic acid, ascorbic acid and acetylglycine can be mentioned. it can.

These peptides of the present invention can be administered orally or parenterally for therapeutic purposes. As the orally-administered agent, solid preparations such as powder, granules, capsules and tablets, or liquid preparations such as syrups and elixirs can be used. In addition, parenteral administration agents such as injections, rectal administration agents, external preparations for skin and inhalants can be used. These formulations are manufactured in a conventional manner by adding to the active ingredient a pharmaceutically acceptable manufacturing auxiliary agent. Further, it is also possible to prepare a sustained-release preparation by a known technique.

To prepare solid preparations for oral administration, active ingredients and excipients such as sugars such as lactose and mannitol, starch, crystalline cellulose, calcium lactate, calcium citrate, magnesium aluminometasilicate, anhydrous silicic acid, etc. To form a powder, or if necessary, add a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, etc., a disintegrating agent such as carboxymethylcellulose, carboxymethylcellulose calcium, etc., and granulate it by wet or dry granulation. Use as an agent. To manufacture tablets, these powders and granules may be tableted as they are or by adding a lubricant such as magnesium stearate and talc. These granules or tablets are coated with an enteric base such as hydroxypropylmethylcellulose phthalate, methacrylic acid, and methyl methacrylate copolymer to form an enteric preparation, or ethylcellulose, carnauba wax, hardened oil, etc. to form a sustained-release preparation. You can also do it. In order to produce capsules, a hard capsule such as powder or granules may be filled, or the active ingredient may be dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and then coated with a gelatin film to give a soft capsule. it can.

To prepare a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol and glycerin are dissolved in water to prepare a transparent syrup, and essential oil, ethanol and the like are added to form an elixir. Alternatively, gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose, etc. may be added to prepare an emulsion or suspension. If desired, a flavoring agent may be added to these liquid formulations.
Coloring agents, preservatives and the like may be added.

In order to produce an injection, an active ingredient may be added, if necessary, to a hydrochloric acid, sodium hydroxide, emulsion, pH adjusting agent such as sodium lactate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, glucose and the like. Dissolve in distilled water for injection with an isotonicity agent, aseptically filter and fill ampoules, or add mannitol, dextrin, cyclodextrin, gelatin, etc. and lyophilize under vacuum to prepare a soluble injection before use. Alternatively, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. may be added to the active ingredient and emulsified in water to give an emulsion for injection.

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

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

To produce an inhalant, the active ingredient is dissolved or dispersed in a propellant such as Freon gas and filled in a pressure resistant container to form an aerosol agent.

The dose of the peptide of the present invention depends on the age of the patient,
Usually about 1 mg to 5 per day, depending on body weight and condition
It is 00 mg, and it is desirable to administer in 1 to several divided doses.

The biological evaluation of this peptide was carried out by measuring the NGF production promoting activity of the peptide on LM cells (see Examples).

Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to these.

[0020]

【Example】

Example 1 Synthesis of Peptide Represented by SEQ ID NO: 1 For the synthesis of peptides, the solid phase method was adopted, and an automatic peptide synthesizer (Model 430A or 431A manufactured by Applied Biosystems) was used according to the program of the company. Carried out. If the reagent used in the synthesizer is 430A, 1. Diisopropylethylamine
2. Trifluoroacetic acid 3. Nutryza- (Ethanolamine) 4. Methanol 5. Hydroxybenzotriazo-
It is 6. Dicyclohexylcarbodiimide 7. Dichloromethane 8. Dimethylformamide. In the case of 431A, N-methylpyrrolidone was used instead of dimethylformamide. The amino acids used in the synthesis were Boc-Ala,
Boc-Arg (Mts), Boc-Asn, Boc-Asp (OBzl), Boc-Glu (OB
zl), Boc-Gln, Boc-Gly, Boc-His (Dnp), Boc-Leu, Boc-
Lys (Cl-Z), Boc-Phe, Boc-Ser (Bzl), Boc-Thr (Bzl),
Boc-Trp (CHO), Boc-Tyr (Br-Z) and Boc-Val. PAM resin was used for the synthesis of C-terminal carvone type peptide, and P-methyl BHA resin was used for the synthesis of C-terminal amide type peptide. Applied Bi is used for the removal of protecting groups and the cutting out from the resin.
According to the manual of osystems. The reagents (peptide resin 1g scale) used for cutting out the peptide resin synthesized using Boc-Trp (CHO) are as follows. 1. low temperature conditions, m-cresol 1 ml, dimethyl sulfide 3 ml, trifluoroacetic acid 5 ml, trifluoromethane sulfonic acid 1 ml, then 2. high temperature conditions, thioanisole: 1,2-ethanedithiol (2: 1) 1.5 m
l, 10 ml of trifluoroacetic acid and 1 ml of trifluoromethanesulfonic acid.

In the synthesis of the peptide of this example, Bo
Method c was adopted and 0.5 mmol Boc-Gly-PAM resin was used. The synthesis was completed and 2.7 g of peptide resin was obtained. Suspending the above peptide resin in dimethylformamide (65 ml),
After adding thiophenol (2.7 ml), the mixture was stirred for 1 hour at room temperature, and first, the protective group Dnp for histidine was removed. The peptide resin was filtered off and washed with dichloromethane. The peptide resin was cut out according to the manual of Applied Biosystems. That is, m-cresol (2.1 ml) and dimethyl sulfide (8.1 ml) were added, and trifluoroacetic acid (13.5 ml) and then trifluoromethanesulfonic acid (2.7 ml) were added to the stirred mixture while cooling in an ice bath. It was Stirred at that temperature for 3 hours. Ether (150 ml) was added and the reaction mixture was filtered,
It was washed 3 times with ether. Thioanisole: 1,2-ethanedithiol (2: 1) (3.0 ml) was added to the obtained mixture,
Stir for 10 minutes. Then trifluoroacetic acid (30 ml) was added while cooling in an ice bath. Trifluoromethanesulfonic acid (2.0 ml) was slowly added, and the mixture was stirred at room temperature for 30 min. Chilled ether was added and the reaction mixture was filtered and washed 3 times with ether.

The crude peptide obtained was treated with trifluoroacetic acid.
It was dissolved in (15 ml), cooled, and added dropwise to the stirred ether. The precipitate was filtered and washed 3 times with ether. It was dissolved in a 10% aqueous acetic acid solution and filtered. This crude peptide was dissolved in an aqueous acetic acid solution and the open column [Bio-Gel P-2]
Was developed with 10% acetic acid aqueous solution. The eluate containing the target substance was freeze-dried to obtain 0.9 g of a white powder. HPLC [O
DS-80TM column (Tosoh, 21.5mmID x 30cm), acetonitrile linear gradient / 0.1% trifluoroacetic acid]
Lyophilization gave a white powder (104 mg). Analytical column [
ODS-80TM column (Tosoh, 4.6mmID x 15cm) acetonitrile linear gradient / 0.1% trifluoroacetic acid]
C was performed to confirm that the purified product was single. Part of the App
477A Protein Sequencer from lied Biosystems,
Amino acid sequence analysis was performed using a 120A PTH analyzer. Also, after hydrolyzing with 6N hydrochloric acid at 110 ° C for 20 hours, use an A-8700 type amino acid analyzer manufactured by IRIKA KOGYO CO., LTD.
As a result of analysis, it was confirmed to have the sequence of SEQ ID NO: 1.

Example 2 Synthesis of Peptide Represented by SEQ ID NO: 2 The peptide synthesis was carried out by the solid phase method using an automated peptide synthesizer (Model 430A or 431A manufactured by Applied Biosystems). It was carried out by. If the reagent used in the synthesizer is 430A, 1. Dipropylamineamine 2. Trifluoroacetic acid 3. Nutrizer
(Ethanolamine) 4. Methanol 5. Hydroxybenzotriazole 6. Dicyclohexylcarbodiimide 7. Dichloromethane 8. Dimethylformamide. For 431A, N instead of dimethylformamide
-Methylpyrrolidone was used. The amino acids used in the synthesis were Boc-Ala, Boc-Arg (Mts), Boc-Asn, Boc-A.
sp (OBzl), Boc-Cys (4-CH3OBzl), Boc-Glu (OBzl), Boc-G
ln, Boc-Gly, Boc-His (Dnp), Boc-Leu, Boc-Lys (Cl-
Z), Boc-Phe, Boc-Ser (Bzl), Boc-Thr (Bzl), Boc-Trp (C
HO), Boc-Tyr (Br-Z) and Boc-Val. PAM resin was used for C-terminal carboxyl group type peptide synthesis, and P-methyl BHA resin was used for C-terminal amide group type peptide synthesis. Applied Bi is used for the removal of protecting groups and the cutting out from the resin.
According to the manual of osystems.

In synthesizing the peptide of this example, Bo
Method c was adopted and 0.5 mmol Boc-Gly-PAM Resin was used.
The synthesis was completed and 2.6 g of peptide resin was obtained. The above peptide resin was suspended in dimethylformamide (50 ml), thiophenol (2.6 ml) was added, and the mixture was stirred at room temperature for 1 hr. First, the protective group Dnp for histidine was removed. The peptide resin was filtered off and washed with dichloromethane. The peptide resin was cut out by a special method according to the manual of Applied Biosystems. That is, m-cresol (2.1 ml) and dimethyl sulfide (7.8 ml) were added, and trifluoroacetic acid (13.0 ml) and then trifluoromethanesulfonic acid (2.6 ml) were added to the stirred mixture while cooling in an ice bath. It was Stirred at that temperature for 3 hours. Ether (150 ml) was added and the reaction mixture was filtered and washed 3 times with ether. Thioanisole in the resulting mixture:
1,2-ethanedithiol (2: 1) (3.9 ml) was added, and the mixture was stirred for 10 minutes. Then trifluoroacetic acid (26 ml) was added while cooling in an ice bath. Trifluoromethanesulfonic acid
(2.6 ml) was added slowly, and the mixture was stirred at room temperature for 30 minutes.
Chilled ether was added and the reaction mixture was filtered and washed 3 times with ether.

The crude peptide obtained was treated with trifluoroacetic acid.
It was dissolved in (15 ml), cooled, and added dropwise to the stirring ether. The precipitate was filtered and washed 3 times with ether.
It was dissolved in a 10% aqueous acetic acid solution and filtered. This crude peptide was dissolved in an aqueous acetic acid solution and developed with a 10% aqueous acetic acid solution using an open column [Bio-Gel P-2]. The eluate containing the target substance was freeze-dried to obtain 1.68 g of a white powder. HPLC [ODS-
It was fractionated by 80TM column (Tosoh, 21.5 mmID x 30 cm), acetonitrile linear gradient / 0.1% trifluoroacetic acid] and freeze-dried to obtain a white powder (500 mg). Analytical column [ODS-80
HPLC was carried out using a TM column (Tosoh, 4.6 mmID x 15 cm) acetonitrile linear gradient / 0.1% trifluoroacetic acid], and it was confirmed that the purified product was single. Applied part
Biosystems 477A Protein Sequencer, 120A
Amino acid sequence analysis was performed using a PTH analyzer. After hydrolysis with 6N hydrochloric acid at 110 ° C for 20 hours,
The analysis was carried out using an I-8700 amino acid analyzer manufactured by Ichikaika. Based on the analysis results, the peptide sequence
u Ser Trp Tyr His Arg Tyr Gln Asn Lys Arg Ala Gly
It was confirmed to have AlaVal Thr Asp Arg Asp Val Thr Gly.

Example 3 The same reaction procedure as in Example 2 was carried out to obtain the peptide represented by SEQ ID NO: 3.

Example 3 NGF production promoting activity of the peptide of the present invention against LM cells 2 × 10 ⁴ cells / ml (0.5% peptone-containing 199 medium) of L
0.2% M cells per well in a 96-well plate
After culturing in ml for 3 days, the medium was exchanged with each peptide solution diluted with 199 medium containing 0.5% BSA, and the cells were further cultured for 24 hours. After the culture was completed, NGF contained in the culture was measured by the EIA method using an anti-mouse NGF polyclonal antibody.
As a result, the peptide dose-dependently promoted NGF production as shown in FIGS. 1 and 2 (the vertical axis represents the NGF production amount and the horizontal axis represents the peptide concentration).

[0029]

[Sequence list]

SEQ ID NO: 1 Sequence length: 26 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide sequence Thr Val Asp Arg Asp Thr Val Ala Gly Ala Arg Lys Asn Gln Tyr 5 10 15 Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Gly 20 25

SEQ ID NO: 2 Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Phe Leu Asn Glu Ser Trp Tyr His Arg Tyr Gln Asn Lys Arg 1 5 10 15 Ala Gly Ala Val Thr Asp Arg Asp Val Thr Gly 20 25

SEQ ID NO: 3 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence

[Brief description of drawings]

FIG. 1 NG of the peptide of SEQ ID NO: 1 in LM cells
It is a graph which shows F production promotion activity.

FIG. 2 is a graph showing NGF production promoting activity of peptides of SEQ ID NOs: 2 and 3 in LM cells.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Akari Sasano 5-4-7 Sagami Ohno, Sagamihara City, Kanagawa Prefecture

Claims (1)

[Claims] 1. A peptide represented by (1) SEQ ID NO: 1 (2) SEQ ID NO: 2 or (3) SEQ ID NO: 3 (wherein the N-terminal amino group of the peptide represented by each SEQ ID NO is acetyl group, t- A nerve growth factor production-promoting agent comprising a butoxycarbonyl group or a benzyloxycarbonyl group, and the C-terminal carboxyl group may be an amide group) as an active ingredient.