JP2005239712A - Neurite outgrowth promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a low molecular weight compound having neurite outgrowth promoting action. <P>SOLUTION: A neurite outgrowth promoter contains a growth hormone liberation accelerating substance as an active agent. A growth hormone liberating peptide is used as the growth hormone liberation accelerating substance in the neurite outgrowth promoter. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a neurite extension agent containing a growth hormone release promoting substance as an active ingredient.

  Nerve cells extend neurites under the influence of the external environment during development, forming a neural circuit. Synapses are formed between the nerve endings and the dendrites of neurons of other neurons, and signal transmission is thereby performed. Since the disappearance of neurites is seen in patients with memory impairment or dementia, synapse formation associated with neurite outgrowth is considered to be closely related to memory. Therefore, by extending the neurites of neurons that have broken neural networks, it is possible to reorganize and enhance synaptic responses between neurons, and to restore functions before being damaged, for example, to improve memory It can be expected to treat dementia.

  It is speculated that neurotrophic factors represented by nerve growth factor (NGF) promote the generation of nerve cells, maintain their survival, and repair the brain when damaged. However, NGF has 118 amino acids, has a large molecular weight (molecular weight 13,259), cannot pass through the blood-brain barrier, and is difficult to move to the brain, and therefore has a problem as a medicine. Therefore, a compound having a low molecular weight and the above activity is required.

  On the other hand, growth hormone release-promoting substances called growth hormone-releasing peptide (hereinafter abbreviated as GHRP) and growth hormone secretion-promoting substance (hereinafter abbreviated as GH secretagogue, GHS) are known to have various physiologically active actions. It has also been reported to have an effect of preventing cell apoptosis and necrosis (Patent Document 1). However, this report aims to prevent the degeneration or death of nerve cells, with the recognition that it is extremely difficult to restore functions lost due to the degeneration or death of neurons and to reconstruct higher brain functions. there were.

International Patent Publication WO01 / 47558

  An object of the present invention is to provide a low molecular compound having a neurite extension action.

  The present inventors have intensively studied a low molecular weight compound that functionally repairs the breakdown of the neural network, and surprisingly found that a growth hormone release promoting substance has a neurite extension action, thereby completing the present invention. It came to.

The present invention provides a neurite extension agent (hereinafter referred to as “the preparation of the present invention”) containing a growth hormone secretagogue as an active ingredient.
According to another aspect of the present invention, there is provided the neurite extension agent, wherein the growth hormone release promoting substance is a growth hormone release peptide.

  According to another aspect of the present invention, there is provided the neurite extender as described above, wherein the growth hormone-free peptide is pralmorelin, ghrelin, a ghrelin analog, or a pharmaceutically acceptable salt thereof.

According to another aspect of the present invention, there is provided the above-mentioned neurite extender, wherein the growth hormone-free peptide is pralmorelin or a pharmaceutically acceptable salt thereof.
According to another aspect of the present invention, there is provided the neurite extension agent which is an agent for improving nerve palsy.

According to another aspect of the present invention, there is provided the neurite extender that is a dementia ameliorating agent.
According to another aspect of the present invention, there is provided the neurite extender that is a memory improving agent.
According to another aspect of the present invention, there is provided the neurite extender which is a brain function activator.

The present invention is described in detail below.
The preparation of the present invention can reorganize and enhance the synaptic response between nerve cells by neurite extension action, and can recover the function reduced by the neurological disease.
Specifically, the following diseases can be cured or symptoms can be improved:
Nerve paralysis associated with peripheral nerve diseases such as amyotrophic lateral sclerosis, diabetic neuropathy, neuropathy, traumatic nerve injury and neurodegeneration, toxic neuropathy, multiple sclerosis (exercise) And improvement of memory impairment in central nervous system disorders and diseases such as Alzheimer's disease, vascular dementia, Parkinson's disease, Huntington's disease, spinal cord injury, treatment of dementia, and brain function activation.

The “growth hormone release promoter” of the present invention includes a growth hormone release peptide (GHRP) and a growth hormone secretion promoter (GH secretagogue, GHS).
“Growth hormone-free peptide (GHRP)” refers to a peptide having a pharmacological activity that promotes growth hormone release. As long as it has such an equivalent function, various derivatives (for example, derivatives in which peptide-constituting amino acids are substituted, Also included are ester derivatives). Especially regarding the number and origin of amino acid residues and amino acid derivative residues in peptides (for example, those isolated and purified from human cells, synthetic products, semi-synthetic products, those obtained by genetic engineering, etc.) There is no.

  Here, the “amino acid derivative” includes alkyl-substituted tryptophan, β-naphthylalanine, α-naphthylalanine, 3,4-dihydrophenylalanine, methylvaline, and the like. Amino acids and amino acid derivatives include both L-form and D-form.

  “Growth hormone secretion-promoting substance” refers to a non-peptide substance having a pharmacological activity that promotes growth hormone secretion, and various derivatives (for example, ester derivatives) are also included as long as they have such an equivalent function.

  Specific compounds of growth hormone-free peptide (GHRP) include, but are not limited to, pralmorelin, hexarelin, GHRP-1, GHRP-6 (SK & F-110679), ghrelin and ghrelin analogs, or their Examples thereof include salts and esters. Preferred examples include pramorelin, ghrelin and ghrelin analogs or salts thereof, and more preferred examples are ghrelin and ghrelin analogs or salts thereof.

Examples of these salts are described below, and preferred examples include hydrochloride (for example, pralmorelin dihydrochloride, hexarelin hydrochloride).
Ghrelin is a peptide having 28 amino acids, represented by the chemical structure described in the table below. Ghrelin analogs include those in which one or more of 28 amino acids are deleted, substituted or added as long as they have a growth hormone release-promoting action. Derivatives in which amino acids are substituted (including those in which a group such as alkylene is inserted between amino acids and ester derivatives) are also included.

  The origin of amino acid residues and amino acid derivative residues in peptides (for example, those isolated and purified from human and rat cells, synthetic products, semi-synthetic products, those obtained by genetic engineering, etc.) are particularly limited. There is no.

Examples of those in which one or more of 28 amino acids are deleted, substituted or added include the following.
A typical example of deletion of one amino acid of ghrelin is des-Gln14-ghrelin, which lacks the 14th Gln residue.

Further, the ghrelin analog of the present invention includes the followings described in J. Med. Chem. 2000, 43, 4370-4376.
For example, among 28 amino acids of ghrelin, the amino acid has 3rd and 4th amino acids from the N-terminus (preferably 4 amino acids at the N-terminus), and the side chain of the 3rd amino acid from the N-terminus (Ser) Peptides and derivatives thereof, which have growth hormone release promoting action.

Examples of the side chain of the third amino acid from the N-terminal include acyl groups and alkyl groups other than octanoyl which is the side chain of ghrelin (the number of carbon atoms is preferably 6 to 18).
Specific examples include:
_{-CH 2 (CH 2) 9 CH} 3, -CO- (CH 2) 6 CH 3, -CO-CH = CH-CH = CH-CH = CH-CH 3, -CO-CH (CH 2 CH 2 CH _{3) 2, -CO- (CH 2} ) 9 CH 3, -CO- (CH 2) 14 CH 3, -CO- (CH 2) 6 CH 2 Br, -CO-CH (CH 2) 2 CONH (CH _{2) 2} CH _3, -COPh,
Following formula:

As a specific example of a ghrelin analog having the 3rd and 4th amino acids from the N-terminus and the side chain of the 3rd amino acid (Ser) from the N-terminus being substituted, the 37th Peptide Discussion Session ( Compounds reported on October 18-20, 2000):
_{NH 2 - (CH 2) 4} -CO-Ser ( _{octyl) -Phe-Leu-NH- (CH} 2) 2 -NH 2 and the like

  Specific compounds of growth hormone secretagogue (GH secretagogue, GHS) include, but are not limited to, butamorelin [eg, butamorelin mesylate (MK-0677)], capromorelin (CP-424391) , Tabimorelin (NN-703) and NNC-260722, or salts and esters thereof. Examples of these salts are described below, and preferred examples include hydrochloride.

  Representative of the above compounds are detailed in the table below.

  Examples of pharmaceutically acceptable salts of the above compounds include salts with mineral acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as methanesulfonic acid, benzenesulfonic acid, malic acid, citric acid, and succinic acid. And salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, salts with basic amino acids such as arginine, and the like.

In the preparation of the present invention, a growth hormone release-promoting substance and a pharmaceutically acceptable salt thereof may be used in combination of two or more.
The growth hormone release-promoting substance used in the present invention can be prepared by a known pharmaceutical technique, alone or in combination with pharmacologically acceptable carriers, additives, etc., with ordinary oral preparations and parenteral preparations such as liquids ( Injectables, nasal drops, syrups, dry syrups, etc.), tablets, troches, capsules (hard, soft, microcapsules, etc.), powders, fine granules, granules, ointments, suppositories, etc. In addition, it can also be formulated into a dosage form such as a drug delivery system (for example, sustained release agent).

  Carriers, additives, and the like that can be used in the preparation of the present invention include those used in the preparation of conventional pharmaceuticals such as the following: for example, physiological saline, water (normal water, distilled water, purified water, injection) Water), aqueous solvents such as Ringer's solution, for example, non-aqueous solvents such as oil-based solvents (such as vegetable oil), and water-soluble solvents (such as propylene glycol, macrogol, ethanol, glycerin), such as cacao butter, polyethylene glycol, micro Bases such as crystallin wax, white beeswax, liquid paraffin, white petrolatum, such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, excipients, cellulose, methylcellulose, hydroxy Propylcellulose, polypropylpyrrolidone, gelatin, ara Binders such as gum arabic, polyethylene glycol, sucrose, starch, etc. Disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, carboxymethylcellulose calcium, calcium citrate, eg magnesium stearate, talc, lauryl Lubricants such as sodium sulfate, such as citric acid, menthol, glycine, sorbitol, orange powder, etc., such as paraoxybenzoates, benzyl alcohol, chlorobutanol, quaternary ammonium salts (benzalkonium chloride, benzethonium chloride) Etc.) and preservatives such as albumin, gelatin, sorbitol, mannitol and the like stabilizers such as methylcellulose, polyvinylpyrrolidone, steari Suspending agents such as aluminum acid, plasticizers such as glycerin and sorbitol, dispersants such as hydroxypropyl methylcellulose, solubilizing agents such as hydrochloric acid and cyclodextrin, emulsifiers such as sodium monostearate, sodium chloride Electrolytes such as sugar alcohols, non-electrolyte osmotic pressure adjusting agents such as sugars and alcohols, and flavoring agents.

  In the case of oral preparations, water-swellable cellulose (carboxymethylcellulose) such as crystalline cellulose (“Avicel” [trade name, manufactured by Asahi Kasei Kogyo Co., Ltd.], etc. Carboxymethylcellulose calcium, sodium carboxymethylcellulose, low-substituted hydroxypropylcellulose, etc.).

  Usually, mammals including humans (for example, mice, rats, hamsters, rabbits, cats, dogs, etc.) by means such as oral, subcutaneous, nasal injection, arterial injection (including infusion), intravenous injection, spinal cord injection, or topical brain administration Cattle, horses, sheep, monkeys, etc.).

  The dosage of the preparation of the present application varies depending on the age, weight, symptoms, administration route, etc. of the patient. For example, when used for the treatment of adults (weight 50 to 70 kg), it is orally administered as a growth hormone release promoting substance. For administration, about 1 mg to 100 mg per kg body weight, and for intravenous administration, about 0.1 to 1000 μg per kg body weight can be administered in 1 to 4 divided doses per day.

  The present invention will be described in more detail with reference to the following examples, but the present invention should not be construed as being limited to these descriptions.

Example 1 (neurite extension effect test)
PC12 cells derived from rat adrenal pheochromocytoma were seeded in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 5% horse serum at ² × 10 ⁵ cells / well on a 25 cm ² plate, The cells were cultured in a 37 ° C. CO ₂ incubator.

Each drug, rat ghrelin (Peptide Institute) and pralmorelin dihydrochloride (KP-102) (Kaken Pharmaceutical Co., Ltd.) was added directly to the above medium to a predetermined concentration using a 10 ⁻⁴ M stock solution. .

  One week after drug treatment, the number of cells having neurites (dendrites and axons) of 1.5 times or more of the total cell number and cell body diameter was counted, and the number of cells having the neurites relative to the total number of cells The percentage was calculated. Next, the ratio of the ratio of the number of cells having neurites to the ratio of the total number of cells to the ratio in the control group was determined, and this is shown as neurite extension in FIG.

Experimental Results In the control group cells, neurite outgrowth was slightly observed after 7 days of culture.
PC12 cells had a weak neurite extension action by NGF, whereas neurite extension action was observed by ghrelin and KP-102.

In any group to which ghrelin (10 ⁻⁹ −10 ⁻⁷ M) was added for 7 days, a remarkable neurite outgrowth effect of 1.5 to 1.9 times was observed at any concentration compared to the control group.
In the group to which KP-102 (10 ⁻¹⁰ −10 ⁻⁹ M) was added for 7 days, a significant neurite outgrowth effect was observed compared to the control group.

Example 2
It was confirmed below that the neurite outgrowth action by the growth hormone release promoting substance in Example 1 is an action via the growth hormone release promoting substance receptor.

Rat ghrelin (Peptide Institute) and pralmorelin dihydrochloride (KP-102) (Kaken Pharmaceutical Co., Ltd.) were used as growth hormone release promoting substances. Rabbit anti-rat GHS-R antibody (Alpha Diagnostic international Inc., Cat #: GHSR11-S, Antiserum), a ghrelin antibody (1000-fold dilution) (hereinafter referred to as “ghrelin antibody”) ), (D-Lys ³ ) -GHRP-6 (Bachem) (hereinafter referred to as “D-Lys”) and (D-Arg ¹ , D-Phe ⁵ , D-Try ^7,9 , Leu ¹¹ )- Substance P (Bachem) (hereinafter referred to as “SubP”) was used.

PC12 cells derived from rat adrenal pheochromocytoma were seeded in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 5% horse serum at ² × 10 ⁵ cells / well on a 25 cm ² plate, The cells were cultured in a 37 ° C. CO ₂ incubator.

The growth hormone release promoting substance and the growth hormone release promoting substance receptor antagonist were directly added to the medium so as to have predetermined concentrations. The final concentrations of ghrelin were 10 ⁻⁸ M, KP-102 was 10 ⁻⁹ M, D-Lys was 10 ⁻⁴ M, and SubP was 10 ⁻⁶ M. When examining the effects of growth hormone release promoting substance receptor antagonists on growth hormone release promoting substances' neurite outgrowth promoting action, growth hormone release promoting substance receptors and growth hormone release promoting substance receptor antagonists are simultaneously applied to the medium. Added.

  When culturing for one week after drug addition, count the number of cells with neurites (dendrites and axons) at least 1.5 times the total cell number and cell body diameter, and the total number of cells with neurites The ratio to the number was calculated. Next, the ratio of the ratio of the number of cells having neurites to the ratio of the total number of cells to the ratio in the control group was determined, and this is shown in FIGS.

Experimental Results No neurite outgrowth effect on PC12 cells was observed in the group (n = 3) to which the growth hormone release promoting substance receptor antagonist alone was added (see FIG. 2).

  In the group (n = 3) to which the antagonist of ghrelin and growth hormone release promoting substance receptor was added, the neurite outgrowth promoting action of the group to which ghrelin alone was added (n = 3) was observed (FIG. 3). reference).

  In the group (n = 3) to which an antagonist of KP-102 and a growth hormone release promoting substance receptor was added, the neurite outgrowth promoting action of the group to which KP-102 alone was added (n = 3) was observed. (See FIG. 4).

  From the above results, it was confirmed that the neurite outgrowth action by the growth hormone release promoting substance was expressed through the growth hormone release promoting substance receptor.

  It has been clarified for the first time by the present invention that a growth hormone release-promoting substance is effective as a neurite extension agent. The preparation of the present invention is an agent for improving nerve palsy (including movement disorders and sensory disturbances) associated with diseases of peripheral nerves, an agent for improving memory disorders in central nerve disorders and diseases, and dementia, due to the neurite outgrowth action. It is useful as a therapeutic agent for cerebral function and a brain function activator.

FIG. 1 shows the effect of ghrelin and KP-102 (pralmorelin dihydrochloride) on neurite outgrowth in PC cells. Values are mean ± S. E. It shows with. *: P <0.05, **: p <0.01 vs. Contrast FIG. 2 shows the neurite outgrowth action of a growth hormone release promoting substance receptor antagonist. FIG. 3 shows the effect of an antagonist of a growth hormone release promoting substance receptor on the neurite outgrowth action of ghrelin. FIG. 4 shows the influence of an antagonist of a growth hormone release promoter receptor on the neurite outgrowth action of KP-102 (Plarmorelin dihydrochloride).

Claims (8)

  Neurite outgrowth agent containing growth hormone release promoting substance as an active ingredient.   The neurite extension agent according to claim 1, wherein the growth hormone release promoting substance is a growth hormone release peptide.   The neurite extender according to claim 2, wherein the growth hormone-free peptide is pralmorelin, ghrelin, a ghrelin analog or a pharmaceutically acceptable salt thereof.   The neurite extender according to claim 2, wherein the growth hormone-releasing peptide is pralmorelin or a pharmaceutically acceptable salt thereof.   The neurite extension agent according to any one of claims 1 to 4, which is an agent for improving nerve paralysis.   It is a dementia improving agent, The neurite extension agent of any one of Claims 1-4.   The neurite extension agent according to any one of claims 1 to 4, which is a memory improving agent.   It is a brain function activator, The neurite extension agent of any one of Claims 1-4.