JP2947657B2 - Nerve growth factor production promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

This invention relates to a nerve growth factor (nerv).
e growthfactor (hereinafter abbreviated as "NGF"). More specifically, the present invention relates to a highly safe nerve growth factor production promoter that is effective for preventing aging of the nervous system, preventing progression of neurological disorders, or improving these diseases.

[0002]

2. Description of the Related Art NGF is used in the peripheral nervous system at the embryonic stage.
Promotes sensory and sympathetic ganglion neuron differentiation and growth
Is a peptide that promotes and elongates neurites,
In addition, for mature sympathetic nerve cells,
Is known to be an essential peptide for maintaining
I have. For example, a young animal may be continuously dosed with anti-NGF antibody.
When the physiological activity of NGF is neutralized by
Significant atrophy or death of ganglion neurons has been observed
(R. Levi-Montalcina et al., Physiological Review,Four
8, 534-569, 1968: H. Thoenen et al., Physiologic.
al Review,60, 1284-1335, 1980). Moreover, this phenomenon
Is irreversible and demonstrates the importance of the physiological role of NGF
Is what you do. Applied research on the action of NGF
The sciatic nerve of an adult rat is severed and
These are connected by a silicon tube, and N
Nervous between GF-injected and non-administered groups
As a result of comparing axonal regeneration, NGF was found to be axon regeneration.
Has also been shown to be effective (Rich et al.,
Experimental Neurology,105, 162-170, 1989).

[0003] On the other hand, the importance of NGF in the central nervous system is also known. For example, by administering NGF into the ventricle of a rat whose nerve tract is projected from the septum to the hippocampus, the septum is administered. Inhibition of degeneration and shedding of cholinergic neurons (F. Hefti, Journal of Neuroscie
nce, 6 , 2155-2162, 1986: LFKromer, Science,
235 , 214-216, 1987: LR Williams et al., Pro
ceedings of National Academy of Sciences US A,
83, 9231-9235, 1986) Improvement of learning and memory ability of aged rats by intracerebroventricular administration of NGF (W. Fisher
Et al., Nature, 329, 65-68, 1987), that the pre-administration of NGF suppresses the delayed cell death of hippocampal pyramidal cells observed after cerebral ischemia (Shigeno et al., History of Medicine, Vol. 145). ,
579-580, 1988).

[0004] As described above, since NGF is an indispensable factor for the survival of nerve cells, a substance which promotes the production of NGF has been searched for the purpose of preventing or treating the progression or treatment of neurological diseases and disorders. I have. As a result, catecholamines (Y. Furukawa et al., Journal of
Biological Chemistry, 261 , 6039-6047, 1986), cholinergic agonists (JP-A-63-83020), cinnamic acid amide compounds (JP-A-2-104568), benzoquinone derivatives (R. Takeuchi et al., FEBS) Letter, 261, 63-6
6, 1990), and propentofylline (I. Shinoda)
Et al., Biochemical Pharmacology, 39 , 1813-1816, 1990
) Is reported to have an NGF production promoting action. Also, fibroblast growth factor (fibroblast growth fa)
ctor), platelet-derived growt
h factor), transforming growth factor alpha and beta
β), epidermal growth factor
), And insulin-like growth factor (insullin-like g)
row growth factor) and other so-called cell growth factors
Has been observed (Shinoda et al., Biochemistry, Vol. 62, p. 835, 1990).

[0005]

With aging, the physiological function gradually decreases without subjective symptoms, and it is difficult to improve the symptoms even if a drug is administered when the body is modulated. Prevention of the so-called physiological aging is one of the most important issues in medical care at present. In order to prevent physiological aging, it is also important to pay close attention to daily dietary habits, and it is desirable to ingest substances that control aging through foods. Ingestion of the substance is expected to be particularly effective in preventing aging of the nervous system.

[0006] In addition, for various neuropathies caused by nerve cell damage, it is considered that the ingestion of such a NGF production promoting substance can be an effective means for preventing or improving the progress of the disorders. Have been. From such a viewpoint, attempts have been made to utilize each of the above-mentioned NGF production promoting substances as pharmaceuticals or foods.

The present invention has been made in view of the above circumstances, and promotes the survival and function maintenance of nerve cells by increasing the production of NGF, thereby preventing or improving aging of the nervous system, and preventing disorders. An object of the present invention is to provide a new highly safe NGF production promoter which prevents degeneration and loss of the cells themselves and prevents or ameliorates the progression of neuropathy.

[0008]

The present invention provides a nerve growth factor production promoter characterized by containing a basic protein or a basic peptide as an active ingredient. I do. Further, the present invention is a substance wherein the basic protein or basic peptide is selected from the group consisting of lactoferrins, lysozyme, ribonuclease A, angiogenin, cytochrome c, lactoferrin N-terminal basic peptide, and a mixture thereof. This is also a preferred embodiment.

In another preferred embodiment, the basic peptide is a basic peptide having in its molecule 2 to 6 peptides having N-terminal and C-terminal basic amino acid residues or a derivative thereof in the molecule. There are.

In the present invention, the lactoferrins are lactoferrin isolated from milk by a conventional method,
Apolactoferrin obtained by removing iron from lactoferrin, and metal-saturated or partially-saturated metal lactoferrin obtained by completely or partially chelating metals such as iron, copper, zinc, and manganese to apolactoferrin. Hereinafter, the NGF production promoter of the present invention will be described in more detail.

The lactoferrin, lysozyme of the present invention,
Ribonuclease A, angiogenin, and cytochrome c are all basic proteins contained in foods such as milk and egg white, and the following physiological effects of each are already known. That is, lactoferrin exhibits an antibacterial effect against harmful microorganisms such as Escherichia coli, Candida, Clostridium, Staphylococci, Enterococci (Jour
nalof Pediatrics, 94, 1, 1979: Journal of Dairy
Science, 67, 606, 1984) that lysozyme has the action of lysing soil bacteria such as Bacillus subtilis, and that ribonuclease A acts on RNA to cut off the phosphate bond between nucleotides and break down the polymerization of molecules. (Iwanami Biological Dictionary, p. 1050, Iwanami Shoten, 1970), Angiogenin is a potent stimulator of angiogenesis (US Pat. No. 4,808,402), and cytochrome c is important for intracellular oxidation (Iwanami Dictionary of Biology, 66
3 pages, Iwanami Shoten, 1970). However, including these substances, other than cell growth factors (the aforementioned fibroblast growth factor, platelet-derived growth factor, and transforming growth factor beta, which react specifically via cell epidermal receptors). Of basic protein is NG
There has been no report of promoting F production.

[0012] The inventors of the present invention searched natural substances for a substance having an NGF production promoting action, particularly from the viewpoint of safety. As a result, these basic proteins and basic peptides were found to have an NGF production promoting action. Has been found. That is, when whey was diluted to a protein concentration of 1 mg / ml and allowed to act on NGF-producing cells, it was found that the amount of NGF produced by the NGF-producing cells increased to about three times that of the control. From this, it was revealed that a substance having NGF production promoting activity was present in whey,
Furthermore, as a result of searching for the carboxymethylcellulose resin-adsorbed fraction of whey to confirm its active substance,
NGF production promoting activity was confirmed for lactoferrin, lysozyme, ribonuclease A, and angiogenin, which are basic proteins. Therefore, lysozyme derived from egg white, which is also a basic protein derived from natural products, ribonuclease A derived from bovine pancreas, angiogenin derived from blood serum, cytochrome c derived from bovine heart, and a basic peptide obtained by degrading lactoferrin As a result of examining certain lactoferrin N-terminal side basic peptides, it was confirmed that these also have NGF production promoting activity.

The present inventors have proposed a basic peptide (Phe-Lys-Cys-Arg-Ar) consisting of 25 amino acid residues at the N-terminal side obtained by hydrolyzing bovine lactoferrin with a protease.
g-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-
Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe. Hereinafter, this peptide is referred to as lactoferricin. Lactoferricin is a trademark owned by the applicant of the present invention), and the short basic peptide constituting this peptide also has an NFG production promoting activity. That is, an N-terminus and a C-terminus are derivatives in which an arginine residue or a lysine residue, respectively, and an arginine residue, a lysine residue or a carboxyl group thereof are substituted with an amide group,
2 with 0-4 optional amino acid residues between them
Up to 6 basic peptides (however, the number of consecutive basic amino acid sequences in the peptide is 2 or less) have NFG production promoting activity.

As shown in the following test examples, iron-saturated transferrin or iron-unsaturated transferrin having a structure similar to lactoferrin (note that these are iron transport proteins like lactoferrin, and the homology between the two is similar) However, lactoferrin contains a large amount of basic amino acids lysine and arginine, and therefore has a large difference in isoelectric point, and transferrin is not a basic protein. ) Were tested for NGF production promoting activity.
As a result, transferrin did not show NGF production promoting activity, confirming the fact that basicity of the protein itself is related to NGF production promoting activity. On the other hand, since there is no difference in NGF production promoting activity between iron-saturated lactoferrin and iron-unsaturated lactoferrin, it was considered that iron was not involved in these NGF production promoting activities. In addition, NGF production promoting activity was also observed on the N-terminal basic peptide of lactoferrin,
It has been found that such basic peptides also have NGF production promoting activity.

The present invention has been made based on the above-mentioned extremely important findings. The NGF production promoter of the present invention can be used for pharmaceuticals or foods. When used as pharmaceuticals, they can be appropriately mixed with excipients, extenders, diluents, solubilizing agents, and the like, and used in the form of tablets, pills, capsules, powders, solutions, suppositories, and the like. . The dose or intake depends on the type of basic protein or basic peptide as the active ingredient, the age of the subject,
It may vary depending on body weight, symptoms, etc.
kg-500 mg / kg is preferable, and the number of amino acid residues is 2-6.
In the case of the basic peptide, it is preferably about 0.02 to 20 mg / kg.

When used for foods, examples thereof include addition to a heated confectionery material, addition to a dressing, use for topping confectionery or cooking, and mixing with sprinkling. In particular, for foods that have been heat-treated for storage or processing or cooking,
Basic proteins such as lactoferrin originally contained therein may be denatured and inactivated by heat treatment. For this reason,
It is extremely nutritionally significant to supplement or enhance the NGF production promoting effect by adding the NGF production promoting agent of the present invention to a heat-treated food material. Of course, it is extremely nutritionally significant to add the NGF production promoter of the present invention to a food that does not originally contain a basic protein such as lactoferrin.

NGF comprising a basic protein or a basic peptide isolated from a natural product such as milk of the present invention as an active ingredient
It is clear that there is no problem in safety as long as an appropriate amount of the production promoter is used. Next, test examples performed to confirm the activity of promoting the growth of nerve growth factor of a basic protein or a basic peptide separated from a natural product are shown below. Test Example 1 (1) Sample preparation Nine kinds of samples shown below were prepared or prepared by the respective methods.

A) Iron-saturated lactoferrin: prepared by the method of Suzuki et al. (Nutrition and Food, Vol. 31, pp. 395-403, 1978). b) Iron-unsaturated lactoferrin: Suzuki et al. (nutrition and food,
31, pp. 395-403, 1978). c) Lactoferrin N-terminal basic peptide: prepared as follows.

50 mg of commercially available bovine LF (manufactured by Sigma) was dissolved in 0.9 ml of purified water, the pH was adjusted to 2.5 with 0.1 M hydrochloric acid, and 1 mg of commercially available pork pepsin (manufactured by Sigma) was added. Hydrolyzed at 6 ° C. for 6 hours. Then pH with 0.1N sodium hydroxide
Was adjusted to 7.0, heated at 80 ° C. for 10 minutes to inactivate the enzyme, cooled to room temperature, and centrifuged at 15,000 rpm for 30 minutes to obtain a clear supernatant. Transfer 100 μl of this supernatant to TSK gel ODS-120T
High-performance liquid chromatography using a Tosoh Corporation (made by Tosoh Corporation) was performed at a flow rate of 0.8 ml / min.
Eluted with 20% acetonitrile, then 0.05% for 30 minutes
Elution was carried out with a gradient of 20-60% acetonitrile containing TFA, fractions eluted between 24-25 minutes were collected and dried in vacuo. This dried product was dissolved in purified water at a concentration of 2% (W / V),
The sample was again subjected to high performance liquid chromatography using TSK gel ODS-120T (manufactured by Tosoh Corporation), eluted with 24% acetonitrile containing 0.05% TFA for 10 minutes after injecting the sample at a flow rate of 0.8 ml / min, and then 0.05% TFA for 30 minutes Was eluted with a gradient of 24-32% acetonitrile containing, and the fractions eluted between 33.5 and 35.5 minutes were collected. The above operation was repeated 25 times and vacuum-dried to obtain about 1.5 mg of the basic peptide.

D) Lysozyme: Commercially available egg white lysozyme (Sigma) is used. e) Ribonuclease A: A commercially available product (derived from bovine pancreas, manufactured by Sigma) is used. f) Angiogenin: prepared by the method described in Japanese Patent Application No. 2-410237. g) Cytochrome c: A commercially available product (from bovine heart, manufactured by Sigma) is used.

H) Iron-saturated transferrin: A commercially available product (manufactured by Yagai Central Research Laboratory) is used. i) Iron-unsaturated transferrin: a commercially available product (manufactured by Yagai Central Research Laboratory) (2) Test method Furukawa et al. (Journal of Biological Chemistry, 261, 6039-
6047, 1986), a mouse connective tissue-derived fibroblast cell line, LM,
Using cells (ATCC, CCL-1, 2), the N
The GF production promoting activity was tested. For culture of LM cells,
A 199 are medium containing 0.5% bactopeptone was used. 5000 cells were seeded per well on a 96-well culture plate and cultured for 24 hours. Next, the culture medium was exchanged with a 199 are culture medium containing the samples (a) to (i) of each concentration shown in Table 1 and 0.5% bovine serum albumin.
Cultured for hours. The LM cells of the control group had no sample added.
The cells were cultured in a 199 medium containing only 0.5% bovine serum albumin. The culture solutions of these test group and control group were collected, and the amount of NGF secreted into the culture solution was quantified and compared.
In addition, for the quantification of NGF, an enzyme immunoassay using an anti-mouse βNGF polyclonal antibody was used. (3) Test results The results are as shown in Table 1. The numerical values shown in Table 1 show the quantitative values of each sample group when the quantitative value of the control group was set to 1.00. As is clear from Table 1, the basic protein or basic peptide of the present invention (Sample Nos. A to g) was significantly NGF-dependent in each addition concentration.
The amount was increased. On the other hand, NGF production promoting activity was not observed in the non-basic protein sample (sample numbers h and i).

[0022]

[Table 1]

Test Example 2 (1) Preparation of Samples Tripeptides at positions 5 to 7 (Arg-Trp-Gln; hereinafter, referred to as sample 1) of lactoferricin (trademark), positions 5 to
A pentapeptide at the 9-position (Arg-Trp-Gln-Trp-Arg; hereinafter, referred to as sample 2);
Peptide substituted with g residue (Arg-Trp-Arg-Trp-Arg; hereinafter, referred to as sample 5); hexapeptide at positions 4 to 9 (Arg-Arg-Trp-Gln-Trp-Arg; hereinafter, sample 3) Described as
A derivative in which the carboxyl group at the C-terminus of this hexapeptide is substituted with an amide group (Arg-Arg-Trp-Gln-Trp-Arg-NH
2. A peptide in which two Arg residues of the pentapeptide were substituted with Lys residues (Lys-Trp-Gln-Trp-Lys; hereinafter referred to as Sample 6).
Shepard et al. [Journal of Chemical Society Parkin I using an automated peptide synthesizer (Pharmacia LKB Biotechnology)
Chemical Society Perkin I), 538, 1981
Year], a solid phase peptide synthesis method.
The test group without additive was used as a control, and the sample of c) of Test Example 1 (hereinafter referred to as Sample 7) was also used for comparison. (2) Test method The same method as in Test Example 1 was used. (3) Test results The results of this test are as shown in FIG. FIG. 1 shows the relationship between the NGF production and the additive concentration of the control and Samples 1 to 6, in which the vertical and horizontal axes represent the NGF production ratio in the medium when the test group without addition was 1, and The concentration of each sample added to the medium is shown, and the number in parentheses shows the sample number (however, sample 7 is not shown). As is clear from FIG. 1, NGF production activity was observed in Samples 2 and 3 having Arg residues as basic amino acids at both ends, and Sample 3 having the amino acid sequence of Arg-Arg was higher than Sample 2. NGF production activity was observed.
Furthermore, Sample 4, which is a derivative of Sample 3 in which the carboxyl group at the C-terminus has been substituted with an amide group, has a lower NGF producing activity expression concentration than Sample 3.

The NGF-producing activity of sample 5 in which the central Gln residue of sample 2 was replaced with an Arg residue was higher than that of sample 2. Furthermore, the NGF-producing activity of Sample 6 in which Arg residues at both ends of Sample 2 were replaced with Lys residues was slightly lower than that of Sample 2, but since NGF-producing activity was observed,
The Lys residue was also found to be effective in expressing NGF production activity. It should be noted that a peptide having an amino acid sequence other than the above samples was also tested, and almost the same results were obtained. Test Example 3 This test was performed to prove the effect of the present invention in an experimental system closer to a living body. (1) Sample preparation a) Lactoferrin: bovine lactoferrin (Yukishira Milk Industry Co., Ltd.)
use. b) Lactoferrin N-terminal basic peptide: prepared in the same manner as in Test Example 1. (2) Test method Ikegami et al. [Biomedic Research
al Research), Vol. 11, p. 61, 1990].

The sciatic nerve of a 5-week-old female SD rat was excised,
Each piece was cultured in a 96-well culture plate using a Dulbecco's modified Eagle's medium (hereinafter abbreviated as FBS-DMEM) containing 10% fetal calf serum. 4
FBS-DMEM containing 100 μg / ml of each sample and FBS-DMEM not containing the sample
(Control), and the medium was changed every day and collected. NGF in the collected culture solution was quantified by an enzyme immunoassay using an anti-mouse βNGF polyclonal antibody. (3) Test results The results of this test are as shown in FIG. -In the figure
-,-▲-, and-△-indicate the control, the sample a, and the sample b, respectively, and the arrow indicates the day on which each sample was added. As is clear from FIG. 2, it was confirmed that when bovine lactoferrin and lactoferrin N-terminal basic peptide were added as compared with the control, NGF production of rat sciatic nerve fragments was significantly promoted. Test Example 4 This test was performed to confirm the NGF-producing effect of a basic peptide having two basic amino acids in the molecule in succession. (1) Preparation of sample Sample 1: Boc-Gln-Gly-Arg-MCA Sample 2: Boc-Gln-Arg-Arg-MCA Sample 3: Boc-Gly-Arg-Arg-MCA Sample 4: Boc-Gly-Lys -Arg-MCA where Boc and MCA denote tertiary butyloxycarbonyl and 4-methyl-cumaryl-7-amide, respectively. The above samples are all commercially available products (manufactured by Peptide Research Laboratories). In addition, the control which does not add a sample was tested similarly. (2) Test method The same method as in Test example 1 was used. (3) Test results The results of this test are as shown in Table 2. As is clear from Table 2, in Sample 1 in which one basic amino acid residue was present in the molecule, no NGF producing effect was observed,
In Samples 2 to 4, in which two basic amino acid residues were present in the molecule, it was found that the amount of NGF produced significantly increased with an increase in the added concentration.

[0026]

[Table 2]

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

[0028]

【Example】

Example 1 (Formulation example) A tablet having the following composition was produced. Iron-saturated lactoferrin 50 mg crystalline cellulose 170 mg corn starch 66 mg talc 11 mg magnesium stearate 3 mg The above ingredients are uniformly mixed, granulated, dried, and tableted.
Tablets were obtained. In addition, iron-saturated lactoferrin was tested in Test Example 1.
It was prepared in the same manner as described above. Example 2 (Formulation example) A capsule having the following composition was prepared.

[0029] Angiogenin 2.0mg Lactose 24.0mg Crystalline cellulose 8.5mg Carboxymethylcellulose 2.0mg Talc 3.0mg Magnesium stearate 0.5mg After the above composition was mixed well, it was made into capsules using a capsule filling machine. In addition, angiogenin was adjusted by the same method as in the test example. Example 3 (Food use example) Japanese confectionery was produced using the following material amounts.

Iron-saturated lactoferrin 50 g Sugar 800 g Kaminami flour 320 g Kanme flour 480 g Shiranami bean paste (60% sugar-containing) 100 g First, put the sugar through a sieve into a container, put the plain bean paste through a sieve sieve, and put it by hand. After kneading, iron-saturated lactoferrin prepared in the same manner as in the test example was added together with Kaminami flour and Kanbai flour, followed by vigorous kneading. This mixture was put in a wooden frame, pressed strongly with a pressing plate, taken out of the wooden frame and air-dried to produce a Japanese confection containing iron-saturated lactoferrin. Example 4 (Formulation example) The following ingredients were uniformly mixed, granulated, dried, and tableted to obtain tablets.

Arg-Arg-Trp-Gln-Trp-Arg 50 mg crystalline cellulose 170 mg corn starch 66 mg talc 11 mg magnesium stearate 3 mg Example 5 The following ingredients are uniformly mixed, filled using a capsule filling machine, and filled into a capsule. I got

Arg-Arg-Trp-Gln-Trp-Arg-NH ₂ 5.0 mg Lactose 21.0 mg Crystalline cellulose 8.5 mg Carboxymethyl cellulose 2.0 mg Talc 3.0 mg Magnesium stearate 0.5 mg

[0033]

As described above in detail, the present invention has the following effects. (1) An NGF production promoter that promotes survival and function maintenance of nerve cells and prevents or ameliorates aging of the nervous system can be obtained. (2) It is possible to obtain an NGF production promoting agent that prevents a degenerated neuron from degeneration and loss of the damaged neuron and prevents or ameliorates the progress of the neuropathy. (3) An NGF production promoter containing a basic protein or a basic peptide separated from a natural product such as milk as an active ingredient,
High security is obtained.

[Brief description of the drawings]

FIG. 1 shows the relationship between the added concentration of various samples and NGF production.

FIG. 2 shows the relationship between the number of days of culture and the amount of NGF in the culture solution.

──────────────────────────────────────────────────の Continuing on the front page (51) Int.Cl. ⁶ Identification symbol FI A61K 38/44 A61K 37/54 38/46 37/43 (72) Inventor Tomoyuki Hagiwara 118 Minami-Kyogaoka, Asahi-ku, Yokohama-shi, Kanagawa Pref. Kibogaoka dormitory (72) Inventor Akira Kaino 118 Minamikibogaoka dormitory, Asahi-ku, Yokohama-shi, Kanagawa Pref. Morinaga Kibogaoka dormitory (56) References JP-A-4-5240 (JP, A) 58) Field surveyed (Int. Cl. ⁶ , DB name) A61K 38/00-38/58 CA (STN)

Claims (1)

(57) [Claims] (1) lactoferrins, lysozyme, ribonucleic acid
Creatase A, angiogenin, cytochrome c, and
Basic protein selected from the group consisting of these mixtures
Quality, or contains an amino acid sequence represented by the following general formula:
It is characterized by containing a basic peptide as an active ingredient.
Nerve growth factor production promoter. X1-X2-X3 (where X1 is an arginine residue or a lysine residue, X2
Is an arbitrary amino acid residue of 0 to 4, X3 is an arginine residue
Amide groups, lysine residues or their carboxyl groups
2 shows a derivative substituted with a group. However, the base in the peptide
Sequence of the amino acid sequence is 2 or less)