JPH07258093A - Agent like nere growth factor - Google Patents

Abstract

PURPOSE:To provide an agent similar to nerve growth factor, containing a sialyl lactose as an active component, effective for promoting the survival of neurocyte and the maintenance of the function, preventing or ameliorating the aging of the nervous system, preventing the degeneration and falling off of damaged neurocyte and preventing or suppressing the progress of nervous disorder. CONSTITUTION:This agent like nerve growth factor contains a sialyl lactose as an active component. The lactose is preferably a 3'-sialyl lactose [O-alpha-D- NeupNAc-(2 3)-O-beta-D-GaLp-(1 4)DGp] of formula I (R is group of formula II) or 6'-sialyl lactose [O-alpha-D-NeupNAc-(2 6)-O-beta-D-GaLp-(1 4)-D-Gp] of formula III (p is pyranose; G is glucose; GaL is galactose; NeuNAc is N-acetyl- neuraminic acid). The compound can be produced e.g. from cow's milk.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a nerve growth factor-like agent. More specifically, the present invention relates to a nerve growth factor-like agent containing sialyllactose as an active ingredient. In the present specification, the representation of constituent sugars means IUPAC except glucose.
(International Union of Pure and Applied Chemistr
Based on the definition of y), the following abbreviations are used and p represents pyranose. G: glucose Gal: galactose GN: glucosamine Neu: neuraminic acid NeuNAc: N-acetylneuraminic acid

[0002]

BACKGROUND OF THE INVENTION Nerve growth factor.
Hereinafter referred to as NGF) is a peptide which promotes differentiation and growth of perceptual and sympathetic ganglion neurons in the peripheral nervous system and promotes neurite outgrowth in the peripheral nervous system, and further matures It is known that sympathetic nerve cells are peptides essential for survival and function maintenance throughout life. For example, when the anti-NGF antibody is continuously administered to juvenile animals to neutralize the physiological activity of NGF,
Significant atrophy of sympathetic ganglia and death of ganglion neurons have been observed [Physiologic Review (Physiolog
ical Review), 48, 534, 1968 and 60, 1284, 1980]. Moreover, this phenomenon is irreversible, demonstrating the importance of the physiological role of NGF.

In addition, application research of NGF is being conducted,
For example, the sciatic nerve of a mature rat was cut, and the sciatic nerve was connected with a silicon tube, and the NGF-administered group in which NGF was injected into the tube and the NGF-non-administered group were compared in terms of the regeneration degree of nerve axons, It has been reported that NGF is also effective for regeneration of nerve axons [experimental
Neurology (Experimental Neurology), 105th
Vol. 162, 1989].

On the other hand, the importance of NGF in the central nervous system is also known. For example, by administering NGF to the ventricle of a rat in which the nerve tract projecting from the septum to the hippocampus has been cut off, Inhibition of degeneration and loss of cholinergic neurons [Journal of Neuroscience, Volume 6, 2155, 1986 and Science, No. 235
Vol. 214, p. 1987], that learning and memory capacity of aged rats are improved by intraventricular administration of NGF [Nature, vol. 329, p. 65, 1].
987], Delayed cell death of hippocampal pyramidal cells after cerebral ischemia is suppressed by pre-administration of NGF [Medical Ayumi, Vol. 145, 579, 1988].
Etc. have been reported.

Thus, since it has been revealed that NGF is extremely important for living organisms, NGF
The search for an NGF-like factor having the same physiological activity as that described above has been promoted. As a result, brain derived neurotrophic factor (BDNF), which is structurally and functionally similar to NGF,
neurotrophin 3 (NT-3), neurotrophin 4 (NT-
4) and neurotrophin 5 (NT-5) were discovered [Nature, Volume 341, Page 149,
1989, Vol. 344, No. 339, 199.
Year 0, Febs Letter (FEBS Letter), Volume 266,
187, 1990 and Neuron,
Volume 6, Page 845, 1991].

Apart from this, NGF which is a structurally different species from NGF
GF-like active substances are also known. Gangliosides are glycosphingolipids containing sialic acid and are constituents of biological membranes [edited by Kazutomo Imahori and Tamio Yamakawa, "Biochemistry Dictionary",
Second edition, page 303, Tokyo Kagaku Dojin, 1990
, It was previously estimated that it plays a role in the nervous system because it exists abundantly in the nervous system, especially in the synaptic region.
F-like neurite outgrowth activity was found [Protein / Nucleic Acid / Enzyme, Vol. 35, pp. 535, 1990], NGF
It is also disclosed that a ganglioside having such physiological activity is applied to a therapeutic drug for Alzheimer-type senile dementia or sequelae of stroke (JP-A-5-271081).

On the other hand, sialyllactose is a general term for sugars consisting of lactose and neuraminic acid, or lactose, neuraminic acid and other monosaccharides. It is an acidic trisaccharide in which N-acetylneuraminic acid is bound to lactose. Certain 3'-sialyllactose (the following chemical formula 1) and 6'-sialyllactose (the following chemical formula 2) are known [Daily Science.
Abstracts (Dairy Science Abstracts), 25th
Volume, No. 11, p. 445, 1963], present in bovine colostrum at a rate of 20-40 mg / 100 ml (supervised by Fujio Egami and Osamu Suzuki, "Polysaccharide Biochemistry-Biology-",
Pp. 1019, Kyoritsu Shuppan, 1970) About 0.03% in oligosaccharides in human milk (Chemistry and Biology, Volume 7, Volume 3)
No., p. 184, 1969) and in human milk 3'-sialyllactose and 6'-sialyllactose are present in a ratio of 85:15 [Archives of Bio. Chemistry and Biophysics,
130, p. 509, 1969].

In addition, di-N-acetyl neuramino.
Lactose [O-α-D-NeupNAc- (2 → 8)
-O-α-D-NeupNAc- (2 → 3) -O-β-
D-Galp- (1 → 4) -D-Gp] (the following chemical formula 3),
Lactopentasaccharide a [O-α-D-NeupN
Ac- (2 → 3) -O-β-D-Galp- (1 → 3)
-O-β-D-GpNAc- (1 → 3) -O-β-D-
Galp- (1 → 4) -D-Gp] (the following chemical formula 4), lacto-pentasaccharide b {O-β-D-Galp- (1
→ 3) -O- [α-D-NeupNAc- (2 → 6)]
-O-β-D-GpNAc- (1 → 3) -O-β-D-
Galp- (1 → 4) -D-Gp} (the following chemical formula 5), lacto-pentasaccharide c {O-α-D-NeupNAc
-(2 → 6) -O-β-D-Galp- (1 → 4) -O
-[Β-α-GpNAc- (1 → 3)]-O-β-D-
It is also known that Galp- (1 → 4) -D-Gp} (the following chemical formula 6) and the like exist in human milk and milk (Taku Mizuno, Kazutoshi Nishizawa, “Glycochemical Chemistry Handbook”, 98-100 pages, Kyoritsu Shuppan, 1971).

[0009]

[Chemical 1]

[0010]

[Chemical 2]

[0011]

[Chemical 3]

[0012]

[Chemical 4]

[0013]

[Chemical 5]

[0014]

[Chemical 6]

However, these sugars (or their hydrolysates) have a bifidobacteria growth-promoting effect, and sialic acid, which is a constituent of sialyllactose, has an anti-inflammatory effect and an infection-protecting effect. (Kunio Yamauchi and Kenkichi Yokoyama, “Milk General Dictionary”, page 519, Asakura Shoten, 1992) and that sialyllactose controls the early immunity of infants (study of dairy science,
(Vol. 17, p. A-55, 1968) is known, but it is not known to have NGF-like activity.

[0016]

Under these circumstances, even if the drug is administered when the physical function is gradually deteriorated due to the aging and physiological function gradually decreases without any subjective symptoms. The prevention of so-called physiological aging, which is difficult to improve the symptoms, has become one of the most important issues in current medical care. In order to prevent physiological aging, it is important to pay sufficient attention to daily eating habits, and it is desired to ingest substances that suppress aging through foods. Ingestion of a substance having physiological activity is expected to be particularly effective in preventing aging of the nervous system.

Also, for various neurological disorders caused by damage of nerve cells, ingestion or administration of NGF-like physiologically active substance can be an effective means for preventing or ameliorating the progression of the disorder. It is estimated that

From the above viewpoints, the present inventors searched for the presence or absence of NGF-like activity in various substances derived from human milk and animal milk in consideration of safety, and found that sialyllact-
The present inventors have found NGF-like activity and have completed the present invention.

The present invention promotes survival and function maintenance of nerve cells to prevent or ameliorate aging of the nervous system, prevents degenerated loss of the damaged nerve cells, and prevents degeneration of nerve cells. It is an object to provide an NGF-like agent that prevents or improves progression.

[0020]

The present invention which solves the above-mentioned problems is a nerve growth factor-like agent characterized by containing sialyllactose as an active ingredient, wherein sialyllactose is 3'- Sialyl lactose [O-α-D-N
eupNAc- (2 → 3) -O-β-D-Galp-
(1 → 4) -D-Gp] and 6′-sialyllactose [O-α-D-NeupNAc- (2 → 6) -O-β-
D-Galp- (1 → 4) -D-Gp] is also a desirable mode.

Next, the present invention will be described in detail. N of the present invention
Sialyl lactose, which is an active ingredient of a GF-like agent, contains sialic acid (neuraminic acid) and lactose as essential components as described above, and may contain other sugars, and contains sialic acid. In terms of points, sialyllactose is clearly a group of compounds distinct from gangliosides, because it is consistent with gangliosides, but since a lipid (ceramide) is not bound to the sugar moiety.

Sialyl lactose can be produced by a known method. An example thereof [Biochimica Biophysica Acta, Vol. 45, p. 601, 1960] is as follows. Degreased milk (colostrum), dialyzed at 5 ° C. for 24 hours, packed with Dowex1-X8, 200-400 mesh (acetic acid type), and washed with water (6.5 × 20c).
The dialysis external solution was passed through m) at a flow rate of 300 to 500 ml / hour, the column was washed with 2 l of water, and the eluate [mixer:
0.085 normal pyridine-acetic acid buffer (pH 4.9-
5.0), leather bar: 0.15 N pyridine-acetate buffer (pH 4.9 to 5.0)] at a flow rate of 10 ml / min, and fractions of 25 ml each are collected. Fractions are collected and lyophilized to give crude sialyllactose.

The lyophilized product was dissolved in methanol to remove insolubles, ether was added to the supernatant, the resulting white precipitate was dried under reduced pressure, and the dried product under reduced pressure was dissolved in water at a ratio of 1 mg / ml. Thoroughly washed Dowex 50-X8, 200-
Pass through a 400-mesh (H ⁺ type) column (1 × 2.5 cm) to remove mixed pyridine, freeze-dry the aqueous solution, dissolve in a small amount of methanol, and precipitate with ether,
The precipitate is dried under reduced pressure to obtain purified sialyllactose.
The yield is 200-400 mg / l bovine colostrum. In addition, it can be produced from bovine colostrum by the method described in Reference Example, and the method of Ogura and Furuhata (Journal of Synthetic Organic Chemistry, Vol. 42, No. 6, 536-543).
(Page, 1984) can also be produced by chemical synthesis. Further, the method of Joziasse et al. [Journal of Biological Chemistry, No. 262]
Vol. 20, page 2025, 1987], it can also be produced from lactose using the enzyme of bovine colostrum (sialyltransferase).

The NGF-like agent of the present invention can be used as a medicine or food. When used as a medicine, it can be used in the form of tablets, pills, capsules, powders, injections, suppositories, etc. by appropriately mixing with excipients, fillers, diluents, solubilizing agents, etc. . The dose or intake of the NGF-like agent of the present invention varies depending on the age, body weight, symptoms, etc. of the subject, but is 0.01 to 100 mg per kg body weight per day.
The degree is desirable. In addition, when it is used for foods, it can be exemplified by adding to confectionery raw materials, toppings for confectionery or dishes, juices, and drinks. Since sialyllactose, which is an active ingredient of the NGF-like agent of the present invention, is a substance obtained from milk, it is clear that it is safe.

Next, the present invention will be specifically described with reference to test examples. Test Example 1 This test was conducted to examine the neurite outgrowth activity of sialyllactose. (1) Preparation of sample a) 3'-sialyllactose [O-α-D-Neup
NAc- (2 → 3) -O-β-D-Galp- (1 →
4) -D-Gp]: commercial product (derived from milk, manufactured by Sigma) b) 6'-sialyllactose [O-α-D-Neup]
NAc- (2 → 6) -O-β-D-Galp- (1 →
4) -D-Gp]: commercial product (from milk, manufactured by Sigma)

(2) Test method Dulbecco's modified Eagle culture medium containing 10% fetal bovine serum (manufactured by Nissui Pharmaceutical Co., Ltd., sometimes referred to as DMEM hereinafter)
24 Neuro2a cells (purchased from Dainippon Pharmaceutical Co., Ltd.) at
Incubated for hours. Then, the culture solution was exchanged with DMEM containing various concentrations of sialyllactose shown in Table 1, and further 24
Incubated for hours. After the culture was completed, the cells were photographed, and the number of protrusions per cell and the elongation of the protrusions were analyzed by an image analyzer MOP-Video Plan (manufactured by Kontron Electronic Co.).

(3) Test Results The results of this test are shown in Table 1. As is clear from Table 1, when the value of the control group without addition is taken as 100%, those of the 3'-sialyllactose and 6'-sialyllactose-added groups have an average concentration of 0.1 μM. The number of protrusions and the average protrusion elongation exceeded 100%, and 3'-sialyllactose and 6'-sialyllactose were found to have neurite outgrowth activity. , Especially in 6'-sialyllactose. In addition, other sialyllactose such as the compounds of Chemical formulas 3 to 6 were also tested, but almost the same results were obtained.

[0028]

[Table 1]

Reference Example 1 The sialyllactose mixture was prepared by the method of Fehre et al.
Journal of chromato
graphy), Volume 212, Page 313, 1981]
Was prepared from colostrum of milk as follows. Chloroform methanol (2: 1, v /
v) add 5 l of solution and stir vigorously at 4 ° C. for 20 minutes,
After centrifugation at 4,000 g for 20 minutes, the upper layer (aqueous layer) was collected and concentrated under reduced pressure. Concentrated liquid (about 300 ml) with Seph
Gel filtration was performed with adex G-25 (fine) resin (manufactured by Pharmacia, column size, 100 × 10 cm) using distilled water at 4 ° C. Glycopeptides and glycolipids elute in the first 650 ml, so the fractions after that (6
Fractions from 50 ml to 950 ml) were collected as sialyllactose fractions. This fraction is Dowex1-X
Sialyl lacto-adsorbed on a resin with 8 mM ion exchange resin (acetic acid type) (manufactured by Bio-Rad), washed with distilled water and adsorbed on the resin with 50 mM pyridine acetate buffer (pH 5.0)
The eluate was lyophilized and the eluate was lyophilized to obtain a sialyllactose mixture (about 800 mg).

Reference Example 2 (Preparation of 3'-sialyllactose and 6'-sialyllactose) From the mixture of sialyllactose obtained in Reference Example 1, the method of Fehner et al. [Journal of・ Chromatography
(Journal of chromatography), Volume 212, Volume 313
Page, 1981] to prepare 3'-sialyllactose and 6'-sialyllactose.

700 mg of sialyllactose mixture was added to 2 parts.
Dowex AG1-X2 ion exchange resin (acetic acid type) dissolved in 80 ml of mM pyridine acetate buffer (pH 5.0) and equilibrated with 2 mM pyridine acetate buffer (pH 5.0) (manufactured by Bio-Rad, column size, 90 x 3c
20 mM pyridine acetate buffer (pH 5.
0) was used to elute at a flow rate of 50 ml per hour, and the eluate was collected in 16 ml aliquots in a test tube. 6'-sialyllactose was added to test tubes Nos. 315 to 340 and 3'-sialyllactose was used. Since the test tubes Nos. 370 to 415 were collected, each was collected and lyophilized to obtain about 400 mg of 3'-sialyllactose and about 40 mg of 6'-sialyllactose.

[0032]

EXAMPLES The present invention will now be described more specifically by showing examples, but the present invention is not limited to the following examples. Example 1 3'-sialyllactose 50 (mg) crystalline cellulose 170 cone starch 66 talc 11 magnesium stearate 3 Each ingredient in the above-mentioned compounding ratio per tablet was uniformly mixed and prepared by a conventional method. Granules were dried, compressed and tablets were obtained. In addition, as the raw materials other than 3'-sialyllactose produced by the same method as in Reference Example 1, commercially available products were used.

Example 2 6'-sialyllactose 5 (mg) lactose 21 crystalline cellulose 8.5 carboxymethylcellulose 2 talc 3 magnesium stearate 0.5 0.5 The mixture was uniformly mixed and filled with a capsule filling machine to obtain a capsule. As the raw materials other than 6'-sialyllactose produced by the same method as Reference Example 2, commercially available products were used.

Example 3 Sialyl lactose mixture 5 (parts by weight) Soft flour 50 Butter 40 Sugar 20 Egg 10 The raw materials were blended in the above proportions, and a sialyllactose-containing cookie was produced by a conventional method. All raw materials other than the -sialyllactose mixture produced by the same method as in Reference Example 1 were commercial products.

[0035]

As described above in detail, the present invention is
The present invention relates to an NGF-like agent, and the present invention has the following effects. (1) A nerve growth factor-like agent that promotes survival and function maintenance of nerve cells and prevents or improves aging of the nervous system can be obtained. (2) NG for a damaged nerve cell, which prevents the degeneration of the cell and prevents or ameliorate the progression of neuropathy
An F-like agent can be obtained.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Norio Ishibashi 5-1-8 Higashihara, Zama City, Kanagawa Prefecture In the Institute of Nutritional Sciences, Morinaga Milk Industry Co., Ltd. (72) Ichizo Shinoda 5-1-8 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd.

Claims (3)

[Claims] 1. A nerve growth factor-like agent comprising sialyllactose as an active ingredient. 2. Sialyl lactose is 3'-sialyllactose [O-α-D-NeupNAc- (2 → 3)].
-O-β-D-Galp- (1 → 4) -D-Gp], The nerve growth factor-like agent according to claim 1. 3. The sialyllactose is 6'-sialyllactose [O-α-D-NeupNAc- (2 → 6)].
-O-β-D-Galp- (1 → 4) -D-Gp], The nerve growth factor-like agent according to claim 1.