阅读说明：本技术 神经生长促进剂 (Nerve growth promoter ) 是由 若山祥夫 田井章博 古贺武尊 于 2018-06-05 设计创作，主要内容包括：本发明的神经生长促进剂含有氨基的氢原子可以经取代基取代的缬氨酸。关于本发明的神经生长促进剂,促进干细胞分化为神经细胞及神经细胞中的神经突起的形成的效果强,并且活性成分不易被消化酶分解。(The nerve growth promoter of the present invention contains valine in which a hydrogen atom of an amino group may be substituted with a substituent. The nerve growth promoter of the present invention has a strong effect of promoting differentiation of stem cells into nerve cells and formation of neurites in nerve cells, and the active ingredient is not easily decomposed by digestive enzymes.)

1. A nerve growth promoter contains valine in which a hydrogen atom of an amino group may be substituted by a substituent.

2. The nerve growth promoter according to claim 1, which further comprises methionine in which a hydrogen atom of an amino group may be substituted with a substituent.

Technical Field

The invention relates to a nerve growth promoter.

Background

Information transmission in the human brain is performed by connecting nerve cells into a mesh-like network via synapses. However, it is said that 10 or more thousands of such nerve cells of an adult disappear every day, and this is one of the causes of central nervous system diseases such as alzheimer dementia. Therefore, it is necessary to identify the cause of such diseases and establish a therapeutic or prophylactic method.

The cause of dementia of the alzheimer type has not been completely elucidated, but there is a tendency to assume that the causative substance is amyloid β or the like. If β amyloid is aggregated in the brain, it is deposited as amyloid spots, and then nerve cells die. Therefore, in many studies to date, corrosion inhibitors against β -secretase or γ -secretase, which are β -amyloid-producing enzymes, or immunotherapy using antigens or antibodies such as amyloid, have been proposed. However, these corrosion inhibitors or therapies have been pointed out to have problems such as insufficient effects or side effects.

On the other hand, studies have also been conducted on the use of neurotrophic factors as therapeutic agents for alzheimer's disease. Neurotrophic factors (neurotropins) are a general term for substances that promote the survival, differentiation, and regeneration of nerve cells, and various trophic factors have been identified (for example, see non-patent document 1). Among them, Nerve Growth Factor (NGF) has been attracting attention as a neurotrophic factor against basalfore-brain cholinergic nerve cells (BFCN) associated with pathological conditions of alzheimer's disease, and has been actively studied (for example, refer to non-patent documents 2 and 3). That is, although alzheimer disease is a dementia disease whose cause is unknown and no effective treatment method is available, BFCN is known to cause significant disorders (reduction in the number of cells, atrophy of cell bodies, and degeneration of neurites) in the brain of the patient. And, BFCN is a nerve conduction pathway related to memory or learning ability, and the dementia pathological condition of alzheimer's disease is highly consistent with a disorder of the nerve conduction pathway. Therefore, as one of the etiologies of alzheimer's disease, a deficiency in NGF is conceivable, and neurotrophic factors are also attempted to be used as therapeutic drugs for alzheimer's disease. For example, when NGF is supplied to a test subject animal in which death of BFCN is induced, the result of inhibition of nerve cell death or the like is obtained (for example, see non-patent document 4 and non-patent document 5). In addition, it has been reported that the number of NGF receptor (TrkA) immunopositive cells in the basal forebrain part of patients with Mild Cognitive Impairment (MCI) or early-stage alzheimer's disease correlates with the performance of cognitive function tests (see non-patent document 6.)

Prior art documents

Non-patent document

Non-patent document 1: drug News Perspectrum, 15,290-

Non-patent document 2: the EMBO Journal,4,1389 (1985)

Non-patent document 3: reviews of Physiology, Biochemistry and Pharmacology,109,145(1987)

Non-patent document 4: neurobiology of Aging,9,689-690(1988)

Non-patent document 5: journal of Neuroscience,5,866(1985)

Non-patent document 6: journal of comprehensive Neurology,427,19-30(2000)

Disclosure of Invention

Technical problem to be solved by the invention

As described above, NGF is attempted to be used as a therapeutic drug for alzheimer's disease. However, since NGF is a high-molecular protein and is decomposed by a protease in the digestive tract, it does not reach the brain even when orally taken. Furthermore, even when administered intravenously, the blood-brain barrier cannot pass through, and therefore, the blood-brain barrier does not reach the brain. Therefore, in order to supply NGF from the outside and to exhibit its nerve differentiation-promoting action or nerve growth action in the brain, NGF can be supplied only directly into the brain parenchyma or the ventricle, which causes a problem of a large burden on the patient.

Therefore, in order to solve the problems of the conventional techniques, the present inventors have studied to provide a drug which has a neural differentiation-promoting action and a neurite-forming action, and in which an active ingredient is not easily decomposed by digestive enzymes and can pass through the blood brain barrier.

Means for solving the technical problem

In order to solve the above problems, the present inventors have focused on amino acids as low molecular weight compounds that can be expected to have physiological activity and enzyme resistance, and examined their effects on nerve cells, and as a result, have found that the compounds are useful as nerve growth promoters, in which neurite outgrowth formation and nerve differentiation-promoting effects are confirmed particularly in valine. The present invention has been made based on these findings, and specifically has the following structure.

[1] A nerve growth promoter contains valine in which a hydrogen atom of an amino group may be substituted by a substituent.

[2] The nerve growth promoter according to [1], which further comprises methionine in which a hydrogen atom of an amino group may be substituted with a substituent.

Effects of the invention

Valine used in the present invention has an action of forming neurites and an action of promoting differentiation of stem cells into nerve cells, and is useful as a nerve growth promoter. Further, valine is a low molecular weight compound, and therefore is not easily decomposed by digestive enzymes and can pass through the blood brain barrier. Therefore, for example, even when a nerve growth promoter containing valine is orally taken, valine can reach the brain while maintaining the structure thereof and exhibit its activity in the brain.

Drawings

FIG. 1 shows Bt representing neurite formation Rate in PC12 cells₂Graph of cAMP concentration dependence.

FIG. 2 shows that Bt was added to an L-valine solution, an L-leucine solution and an L-isoleucine solution at 0.02. mu.g/mL or 0.2. mu.g/mL, respectively₂A graph showing neurite outgrowth rates of cAMP in RPMI-1640 medium of PC12 cells.

FIG. 3 shows the addition of 0.02. mu.g/mL of each of L-valine solution, L-leucine solution and L-isoleucine solution to Bt₂A graph showing neurite outgrowth rates of cAMP in Nutrient Mixture (Nutrient Mixture) F-12Ham medium of PC12 cells in culture.

FIG. 4 shows the addition of Bt to a solution containing 0.2. mu.M or 2. mu.M L-valine and N-acetyl-L-valine₂A graph showing neurite outgrowth rates of cAMP in RPMI-1640 medium of PC12 cells.

FIG. 5 shows the addition of Bt to a solution containing L-methionine and D-methionine at 0.2. mu.M, 2. mu.M or 20. mu.M, respectively₂A graph showing neurite outgrowth rates of cAMP in RPMI-1640 medium of PC12 cells.

FIG. 6 shows the addition of Bt to a solution containing 0.2. mu.M of each of L-valine solution, L-methionine solution and a mixture of L-valine and L-methionine₂A graph showing neurite outgrowth rates of cAMP in RPMI-1640 medium of PC12 cells.

FIG. 7 is a photograph showing a phase contrast and a fluorescence staining image of PC12 cells (control sample 1) cultured without adding NGF and L-valine to the medium, PC12 cells (control sample 2) cultured with NGF added to the medium, and PC12 cells (sample 1) cultured with NGF and L-valine added to the medium.

Detailed Description

The present invention will be described in detail below. The following description of the constituent elements is based on representative embodiments and specific examples, but the present invention is not limited to these embodiments. In the present specification, the numerical range expressed by the term "to" means a range including numerical values before and after the term "to" as a lower limit value and an upper limit value.

The nerve growth promoter of the present invention is characterized by valine in which a hydrogen atom of an amino group may be substituted by a substituent. In the following description, valine in which a hydrogen atom of an amino group is substituted with a substituent may be referred to as "N-substituted valine", and N-substituted valine and unsubstituted valine may be collectively referred to as "N-substituted or unsubstituted valine".

Valine is a compound represented by the following formula, is an α -amino acid in which an amino group is bonded to a carbon at the α -position, and is a Branched Chain Amino Acid (BCAA) having a branched structure.

[ chemical formula 1]

Valine, which is one of the essential amino acids, is known to be used as an energy source in vivo, and has the effects of promoting protein synthesis in skeletal muscle and inhibiting protein degradation. On the other hand, the present inventors have further studied the physiological activity of valine, and as a result, have confirmed that valine has an excellent function as a nerve growth promoter by confirming the action of promoting differentiation of stem cells into nerve cells (nerve differentiation-promoting action) and the action of forming neurites (neurite-forming action). Further, human digestive enzymes are roughly classified into carbohydrases, proteolytic enzymes and lipolytic enzymes, but valine is not decomposed by any of the enzymes and is absorbed in the intestinal tract in its original structure. Also, valine, which is absorbed by the intestinal tract and enters the blood, can cross the blood brain barrier. Therefore, for example, even when a nerve growth promoter containing valine is orally taken, valine can reach the brain in a state where the structure is maintained and its activity can be effectively expressed in the brain.

The N-substituted or unsubstituted valine used in the present invention is preferably a free amino acid. When valine is present as a structural unit of a protein or a peptide, valine is present in a form in which an amino group thereof is subjected to dehydration condensation with a carboxyl group of another amino acid to form a peptide bond, and the carboxyl group thereof is further subjected to dehydration condensation with an amino group of another amino acid to form a peptide bond.

The N-substituted or unsubstituted valine may be in the L form or in the D form, or may be contained in the nerve growth promoter as a mixture of the L form and the D form, but it is preferable that more than 50 mol% of the total amount of the N-substituted or unsubstituted valine contained in the nerve growth promoter is in the L form, more preferably more than 70 mol% is in the L form, still more preferably more than 80 mol% is in the L form, and most preferably all of the N-substituted or unsubstituted valine contained in the nerve growth promoter is in the L form.

The N-substituted or unsubstituted valine used in the present invention may be a synthetic valine or a valine extracted from natural substances. Examples of the method for synthesizing valine include a method in which ammonia acts on α -bromoisovaleric acid, a synthesis method using a strecker reaction, and a synthesis method based on amino acid fermentation using a microorganism. Further, the extraction of valine from natural products can be performed by mechanical disruption or homogenization of natural products, hydrolysis with enzymes, acids, or bases, various separation and purification methods, and the like.

In the N-substituted valine of the present invention, the hydrogen atom substituted with the substituent may be 1 or 2 of 2 hydrogen atoms constituting the amino group, but is preferably 1. When 2 hydrogen atoms of the amino group are substituted with a substituent, the 2 substituents may be the same or different. The type of the substituent is not particularly limited, and examples thereof include an alkyl group and an acyl group. The alkyl group preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and can be selected from the range of 1 to 3 carbon atoms, for example. The acyl group preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and can be selected from the range of 1 to 3 carbon atoms, and acetyl group and the like can be used.

Specific examples of valine in which a hydrogen atom of an amino group is substituted with a substituent (N-substituted valine) are shown below. However, the N-substituted valine that can be used in the present invention should not be construed restrictively by this specific example.

[ chemical formula 2]

N-acetyl-L-valine

The valine in the nerve growth promoter of the present invention may be composed of only unsubstituted valine, or only N-substituted valine, or may include both unsubstituted valine and N-substituted valine. When the nerve growth promoter contains N-substituted valine, the N-substituted valine may be the same or two or more kinds may be combined.

The nerve growth promoter of the present invention may be composed of N-substituted or unsubstituted valine alone, or may contain a component (other component) other than N-substituted or unsubstituted valine. Methionine is a preferred example of the other component that can be used as a nerve growth promoter. By using N-substituted or unsubstituted valine in combination with methionine, the nerve growth promoting effect can be enhanced as compared with the case of using only N-substituted or unsubstituted valine. Methionine is a compound represented by the following formula, and may be an L-form or D-form, or may be contained in the nerve growth promoter as a mixture of the L-form and the D-form.

[ chemical formula 3]

Further, methionine used in combination with N-substituted or unsubstituted valine may be methionine in which a hydrogen atom of an amino group is substituted with a substituent, but is preferably unsubstituted from the viewpoint of enhancing a nerve growth promoting effect, and more preferably, unsubstituted methionine is used in combination with unsubstituted valine. Among them, in methionine in which an amino group is substituted with a substituent (hereinafter, sometimes referred to as "N-substituted methionine"), the hydrogen atom substituted with a substituent may be 1 or 2 of 2 hydrogen atoms constituting an amino group, but is preferably 1. When 2 hydrogen atoms of the amino group are substituted with a substituent, the 2 substituents may be the same or different. As for preferable ranges and specific examples of the substituents, reference can be made to the preferable ranges and specific examples of the substituents in which N is substituted for a hydrogen atom of a substituted amino group in valine described above.

Specific examples of methionine in which a hydrogen atom of an amino group is substituted with a substituent (N-substituted methionine) are shown below. However, the N-substituted methionine that can be used in the present invention should not be construed restrictively by this specific example.

[ chemical formula 4]

N-acetyl-L-methionine

In the case where the nerve growth promoter of the present invention contains valine and methionine (in any case, the hydrogen atom of the amino group may be substituted with a substituent), the content of valine is preferably 1% by weight or more, more preferably 10% by weight or more, and still more preferably 30% by weight or more, relative to the total amount of valine and methionine. Further, it is preferably 99% by weight or less, more preferably 90% by weight or less, and further preferably 70% by weight or less.

Also, for example, in the case of extracting N-substituted or unsubstituted valine from a natural substance, the nerve growth promoter of the present invention may contain a component derived from the natural substance in addition to the N-substituted or unsubstituted valine. The nerve growth promoter of the present invention may contain various components in addition to N-substituted or unsubstituted valine. For example, when an excipient is contained in the nerve growth promoting agent, molding becomes easy, and the nerve growth promoting agent can be increased without changing the content of N-substituted or unsubstituted valine to improve handling properties. The excipient is not particularly limited, but dextrin is preferable. The dilution ratio by the excipient is preferably 2 to 10 times, more preferably 2 to 7 times, and further preferably 3 to 5 times, in terms of mass ratio.

In addition, in the case where the nerve growth promoter contains a component other than N-substituted or unsubstituted valine, the content of N-substituted or unsubstituted valine in the nerve growth promoter is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and still more preferably 1% by mass or more, relative to the total mass of the nerve growth promoter. For example, the content may be 50% by mass or more, 90% by mass or more, or 99% by mass or more.

The nerve growth promoter of the present invention has an action of promoting differentiation of stem cells into nerve cells (nerve differentiation-promoting action) and an action of forming neurites in nerve cells (neurite-forming action), and particularly can effectively promote formation of dibutyryl cAMP (Bt)₂cAMP：Dibutyryladenosine 3’,5’-cyclic monophosphate (dibutyryladenosine 3',5' -monophosphate)) induced neurite outgrowth formation and nerve differentiation induced by Nerve Growth Factor (NGF).

Therefore, when the nerve growth promoter of the present invention is orally taken and the components thereof are absorbed into the intestinal tract, differentiation of stem cells into nerve cells and formation of neurites are effectively promoted in the arriving nervous system, both peripherally and centrally, and the nerve growth promoter contributes to the reconstruction of a nerve circuit damaged by degeneration or damage of the neurites. This enables effective relief of cognitive or motor dysfunction caused by neurodegenerative diseases or nerve injury. The nerve growth promoter disclosed by the invention has the following advantages: since valine as a biological component or N-substituted valine as a valine inducer is used, safety is high, and since valine and most of N-substituted valine are not decomposed by digestive enzymes, it is easy to use as an internal preparation or the like to be taken orally.

The nerve growth promoter of the present invention has an effect of promoting differentiation of stem cells cultured in a medium into nerve cells. Therefore, the nerve growth promoter of the present invention can be effectively used as a differentiation promoter for promoting differentiation of pluripotent stem cells such as iPS cells into nerve cells in the field of regenerative medicine using these stem cells and neural precursor cells. As a result, neural cells can be efficiently produced from stem cells, and a great contribution can be made to the improvement of production efficiency and cost reduction of various industries involved in regenerative medicine.

The amount of the nerve growth promoter of the present invention to be used varies depending on the disorder to be treated, but is preferably used in the following amounts, for example.

For example, when the nerve growth promoter of the present invention is orally administered as an oral drug, the amount of administration is preferably 80 to 2000mg per adult standard body weight per day, and is preferably administered 2 to 3 times per 1 day.

When the nerve growth promoter of the present invention is added to a medium for culturing pluripotent stem cells and neural precursor cells, the amount of the addition is preferably 0.1% by mass or more, more preferably 0.2% by mass or more, and still more preferably 0.2 to 1.0% by mass, based on the total mass. The amount of N-substituted or unsubstituted valine added is preferably 0.03 mass% or more, more preferably 0.05 mass% or more, and still more preferably 0.05 to 0.25 mass% in terms of dry weight.

[ use of nerve growth promoter ]

As described above, the nerve growth promoter of the present invention has a nerve growth promoting effect and also has an effect of promoting differentiation of stem cells such as pluripotent stem cells or neural precursor cells into nerve cells.

Therefore, the nerve growth promoter of the present invention can be effectively used as an internal preparation for alleviating dysfunction caused by neurodegenerative diseases or nerve damage in animals such as humans by supplying it to the animals. The nerve growth promoter as an internal preparation may contain various components in addition to the above decomposition product and excipient as necessary. For example, vitamins, vegetable powder, minerals, yeast extract, coloring agents, thickeners, and the like can be contained as necessary. The kind of these components is not particularly limited, and the content is appropriately adjusted within a range that can sufficiently exhibit the intended function.

In the field of regenerative medicine using pluripotent stem cells such as iPS cells and neural precursor cells, the nerve growth promoter of the present invention can be added to a culture medium or a diluted solution of cells and preferably used as a differentiation promoter for promoting differentiation of these stem cells into nerve cells. The medium to which the nerve growth promoter is added may be any of a liquid (broth) medium, a semi-fluid medium, and a solid (agar) medium, and the composition thereof is not particularly limited. The diluent can be applied to any one of the diluents generally used as a diluent for cells such as physiological saline.