JPH09151132A - Medicine for enhancing action of cell differentiation-inducting factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicine for enhancing the action of a cell differentiation-inducing factor, having e.g. a strong BMP action-enhancing activity, a bone formation-promoting activity and a neurotrophic factor-enhancing activity, and capable of being used for treating and preventing various bone diseases or neuropathies. SOLUTION: This medicine for enhancing the action of a cell differentiation- inducing factor also as a bone disease or neuropathy-treating or preventing composition or as a bone formation-promoting medicine contains helioxanithin or its lactone-opened product or its salt (hereinafter referred to as helioxanthin, etc.). It is also defined as a composition containing helioxanthin, etc., and a cell differentiation-inducing factor or that using helioxanthin, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to treatment / prevention of bone diseases such as osteoporosis and bone fracture, bone reconstruction, or Alzheimer's disease, cerebrovascular dementia, amyotrophic lateral sclerosis (Low-Gerlic disease), and diabetes. The present invention relates to an agent for enhancing the action of a cell differentiation inducing factor, which is effective for the treatment / prevention of neurological diseases such as sexual peripheral neuropathy.

[0002]

2. Description of the Related Art Bone morphogenetic prote
in, BMP) is the only group of protein factors isolated from demineralized bone and known to have ectopic osteoinduction.
Therefore, it is useful as a bone formation promoter in bone fracture healing and bone reconstruction [AE Wang, Trends Biotechnol., Vol. 11, 379-383.
P. (1993)]. Moreover, since BMP directly promotes the differentiation of osteoblasts, BMP is expected to play a role as a coupling factor in bone remodeling, and is considered to be closely related to bone metabolism. In addition, it has been reported that the BMP content in the bone matrix of aged animals is considerably reduced [ML Urist, Bone and Mineral Research, Volume 6 (ed by W.
A. Peck), pp. 57-112, Elsevier, 1989], for maintaining bone mass.
BMP is considered to be deeply involved. This is the BMP
Indicates that it is promising as a therapeutic drug for various bone diseases such as osteoporosis. However, BMP is usually present in a very small amount in the living body, and its source is limited, and BMP is a protein, so there are problems when it is administered, and the applicable target diseases are very limited. .

Furthermore, BMP has been reported to have a neurotrophic factor-like activity [VM Paralkar et al., Journal of Cell Biology, 1].
19: 1721-1728 (1992)]. In addition, strong expression of BMP gene is known in brain tissue [E. Ozkaynak et al., Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Commu
n.), 179, 116-123 (1991)]. It has also been suggested that BMP plays an important role in neural tube formation during embryogenesis [K. Basler et al., Cell, Vol. 73, 687-7].
02 (1993)]. Therefore, it is considered that BMP is deeply involved in the differentiation or function maintenance of nerve cells.

[0004] Neurotrophic factor is a group of protein factors that play an important role in survival maintenance and function expression of nerve cells, and nerve growth factor (nerv
e growth factor (NGF), brain-derived neurotrophic factor (brain-
There are derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). NGF
Is a sympathetic ganglion cell (sympathe) of the neural crest in the peripheral nervous system.
tic ganglion) and dorsal roo ganglion cells (dorsal roo)
(AM Davies & R.
M. Lindsay, Developmental Biology (D
ev. Biol.), 111, 62-72 (1985); R. Levi-Monta.
lcini, EMBO J., 6 volumes, 1145-115
4 (1987)], in the central nervous system, the cholinergic neurons of the septal area (basal forebrain).
a) [H. Gnahn et al., Developmental
Brain Research (Dev. Brain. Res.), Volume 9, 45-52
Page (1983); H. Hatanaka & H. Tsukui, Dev. Brain Re
s., 30, 47-56 (1986); F. Hefti, Journal of Neuroscience (J. Neurosci.), 6, 2155-2.
162 (1986)]. NGF is required to maintain nerve function even after nerve cell differentiation is completed. BDNF acts on dorsal root ganglion cells and nodose ganglion cells in the peripheral nervous system, but not on sympathetic ganglion cells [RM Li
ndsay & H. Rohrer, Dev. Biol., 112, 30-48 (198
5); RM Lindsay et al., Dev. Biol., 112, 319-328.
(1985); AM Davies et al., J. Neurosci., Volume 6, 1897-.
1904 (1986)]. On the other hand, in the central nervous system, cholinergic neurons and GABA (γ-aminobutyric ac
id) Acting nerve cells and dopaminergic neurons of the midbrain (dopaminergic neurons of midbrain) [RF Alderson et al., Neurons (Neuron), Volume 5,
297-306 (1990); C. Hyman et al., Nature (Natur
e), 350, pp 230-232 (1991); B. Knusel et al., Proceeding of the National Academy of Sciences of the United States of America (Proc. Natl. . Acad. Sci. USA), 88
Vol. 961-965 (1991)]. NT-3 is NGF in the peripheral nervous system
And BDNF, but has a strong effect on sensory nerve cells derived from neural plate (P. Ernfors et al., Proc. Natl.
Acad. Sci. USA, 87, 5454-5458 (1990); A. Rosen
Thal et al., Neuron, 4, 767-773 (1990)]. But N
Central nervous system neurons that respond to T-3 are not yet known.

In Alzheimer's dementia, in addition to degeneration and loss of cholinergic nerve cells in the basal forebrain including the septal area, a wide range of damages and loss of cerebral cortical nerve cells have been observed. New trophic factors are also considered as one of the therapeutic agents [F. Hefti & WJ Weiner, Annual Neurology (Annu. Neurol.), Volume 20, 275-281.
P. (1986)]. In addition, in Parkinson's disease, which is a disease in which the midbrain dopaminergic nerve cells of the brain are degenerated and lost, BDNF, which is a trophic factor for the nerve cells, is expected as a therapeutic drug. However, since these neurotrophic factors are proteins, their application is limited.

[0006]

In view of the above points, for example, a compound that enhances the action of BMP can enhance the action of BMP existing in the living body or BMP administered to the living body. It is useful as a therapeutic drug for bone diseases such as. Substances having such BMP action enhancing activity include retinoic acid and vitamins, which have been reported so far.
There are D3, estrogens, and glucocorticoids [V. Rosen & RS Thies, Trends Genet., Vol. 8, pp. 97-102 (1992);
Y. Takuwa et al., Biochemical and Biophysical Research Communication (Biochem. Bioph
ys. Res. Commum.), 174, 96-101 (1991)]. However, when these substances are administered into the body, side effects such as promotion of bone resorption and occurrence of hypercalcemia and ovarian cancer are known, and they are not necessarily suitable as therapeutic agents for bone diseases.

[0007] On the other hand, for example, a compound that enhances the action of NGF can enhance the action of NGF existing in the living body or NGF administered to the living body. It is useful as a medicine. As a substance having such an action, saberuzole (sabeluzole,
4- (2-Benzothiazolylmethl-amino) -α [(p-fluoropheno
[xy)] methyl] -1-piperidineethanol] has been reported [New Current, Vol. 4, No. 26, p. 14]
(1993)], but its mechanism of action has not yet been elucidated, and side effects such as headache, dizziness and tiredness have been observed in clinical trials, and it is not always suitable as a therapeutic drug for neurological diseases. In addition, steroids, catechols and cytokines have been reported as compounds having an NGF secretion-inducing action [Experimental Neurology, Vol. 124, 36-
42 (1993)], Japanese Patent Laid-Open No. 3-81218 has a report on idebenone. However, these compounds are those having neurotoxicity, weakened immunity, hypercalcemia,
Some of them have unfavorable effects such as promotion of bone resorption, and the fact that the NGF secretion-inducing effect and adverse effects on tissues other than the nervous system cannot always be separated, and they are not sufficiently satisfactory for practical use. Furthermore, since the cell differentiation inducing factor represented by BMP or neurotrophic factor is a protein, there is a limit in its administration to the living body. Therefore, as the compound that enhances the action of the cell differentiation inducer existing in the living body or the cell differentiation inducer administered in the living body, a low molecular weight compound is preferable. Therefore, the present invention finds a low molecular weight compound that enhances the action of a cell differentiation inducer represented by BMP or neurotrophic factor, and acts as a cell differentiation inducer useful for the treatment and prevention of various bone diseases or neurological diseases. It is intended to provide a potentiator.

[0008]

[Means for Solving the Problems] Under the technical background, the present inventors aim to develop a drug that specifically enhances the differentiation of osteoblasts and nerve cells by BMP and neurotrophic factors, As a result of intensive research to find a low-molecular compound that enhances the action of a cell differentiation inducing factor,
For the first time, they found that they have excellent activity to enhance the actions of BMP and neurotrophic factors, and completed the present invention.

That is, the present invention relates to helioxanthin,
Alternatively, it is a cell differentiation inducing factor action-enhancing agent comprising a lactone cleavage product or a salt thereof. As described in detail below, the cell differentiation inducing factor is preferably a bone morphogenetic factor, a neurotrophic factor, particularly preferably a neurotrophitic factor family. Also, the present invention
Also provided is a composition for treating / preventing a bone disease or a neurodegenerative disease, which comprises helioxanthin, a lactone cleaved product thereof, or a salt thereof. Furthermore, the present invention also provides a composition comprising helioxanthin, a lactone cleavage product thereof or a salt thereof, and a cell differentiation inducing factor. The present invention also provides a method for enhancing the action of a cell differentiation inducing factor, which comprises using helioxanthin, a lactone cleavage product thereof, or a salt thereof. further,
The present invention also provides an osteogenesis promoter containing helioxanthin, a lactone cleaved product thereof, or a salt thereof.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The lignan derivative used in the present invention, helioxanthin: 1- (3,4-meth
ylene-dioxyphenyl) -2-hydroxymethyl-7,8-methylenedi
[oxy-3-naphthoic acid lactone] is a known compound first identified as a component of plants of the Asteraceae family [RS Bu
rden et al., Journal of the Chemical Society, Section C (J. Chem. Soc. (C)), 693-701 (196).
9)]. Helioxanthin has also been isolated from a plant belonging to the genus Amblyceae known as a crude drug [JM Truji
llo et al., Phytochemistry, 29 ,
2991-2993 (1990)]. The structure of helioxanthin is represented by the following formula (I), and is structurally completely different from the above-mentioned substance having the activity enhancing activity of BMP or NGF.

[0011]

Embedded image

Regarding the chemical synthesis method of helioxanthin, Journal of Natural Products [J. of Natural Product, 52 (2), 367-375 (1989)
Year)]. The lactone cleaved form of helioxanthin used in the present invention has the formula (II):

[0013]

Embedded image

It can be produced by hydrolyzing the lactone portion of helioxanthin by a method known per se to form a carboxyl group and a hydroxyl group.

The salt of the cleaved lactone (II) is preferably a physiologically acceptable salt, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, Examples thereof include salts with basic or acidic amino acids. Preferred examples of the salt with an inorganic base include, for example, sodium salt,
Examples thereof include alkali metal salts such as potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and aluminum salts. Preferable examples of salts with organic bases include ammonium salts, salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N'-dibenzylethylenediamine, etc. Is mentioned. Preferred examples of the salt with an inorganic acid include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Suitable examples of salts with organic acids include, for example, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p -Salts with toluenesulfonic acid and the like. Preferred examples of the salt with a basic amino acid include, for example, salts with arginine, lysine, ornithine, and preferred examples of the salt with an acidic amino acid include, for example, salts with aspartic acid, glutamic acid, and the like. Can be These salts can be obtained according to a conventional method.

The cell differentiation inducer of the present invention includes bone morphogenetic factor, neurotrophic factor, tumor growth factor (TGF)-
Factors belonging to the TGF-β superfamily such as β or activin, factors belonging to the FGF superfamily such as basic fibroblast growth factor (bFGF) or acidic fibroblast growth factor (aFGF), leukemia inhibito
ry factor; LIF, or cholinergic differentiation
factor; sometimes called CDF) or serially neurotrophi factor
c factor; CNTF) and other neuropoietic cytokine famils
y), interleukin-1 (IL-1, abbreviated below), IL-2, IL-3, IL-5, IL-6, IL-7, IL-9, IL-11,
Tumor necrosis factor-α (TNF-α), interferon-γ (INF-
(γ) such as osteoblasts and nerve cells, factors that induce traits characteristic of the process of differentiation of cells that maintain biological functions in specific tissues from undifferentiated precursors, and preferably osteogenic factors Or a neurotrophic factor is mentioned. Bone morphogenetic proteins such as BMP-2, -4, -5, -6-, -7, -8, -9, -10, -11, -12, which are proteins that promote bone formation and cartilage formation The BMP family, especially BMP-2, -4, -6-,-7. The BMP may be a homodimer of each of the factors listed above or a heterodimer with all possible combinations. As neurotrophic factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), glial-derived neurotrophic factor Factor (glia-derived neurotrophic facto
r: GDNF), NT-4 / 5 and the like, preferably the NGF family.

The agent for enhancing the action of a cell differentiation inducer comprising helioxanthin of the present invention is used alone or in combination with a substance having a cell differentiation inducer action, such as BMP or a neurotrophic factor, to promote the healing of bone fractures, bone Reconstruction promotion, treatment and prevention of various bone diseases such as osteoporosis, neurodegenerative disease in cerebrovascular dementia, senile dementia or Alzheimer's disease, amyotrophic lateral sclerosis (Low-Gerlic disease) or It can be used for the treatment and prevention of various cerebral dysfunctions or neurological diseases such as diabetic peripheral neuropathy (neuropathy). The agent for enhancing the action of a cell differentiation inducing factor of the present invention can also be used as a therapeutic or prophylactic agent for such diseases involving BMP, neurotrophic factor and the like. The agent for enhancing the action of a cell differentiation inducing factor of the present invention includes humans as well as other mammals (eg, mouse,
(Rat, rabbit, dog, cat, cow, pig, etc.) can also be applied to the above diseases.

When the agent for enhancing the action of a cell differentiation inducer of the present invention is administered to humans, the administration method may be either oral or parenteral. The agent for enhancing the action of a cell differentiation inducing factor in the present invention can be produced according to a known means for producing a pharmaceutical preparation, except that helioxanthin or a lactone cleaved product thereof or a salt thereof is contained.
In the present invention, helioxanthin or its cleaved lactone or its salt is used alone or in combination with a physiologically acceptable carrier. When compounded with a carrier, the content of helioxanthin or its lactone cleavage product or its salt may be appropriately selected depending on the preparation. The content of helioxanthin or its lactone cleavage product or its salt in the preparation of the present invention is usually about 0.3 to 1
It is 00% by weight, preferably about 0.5 to 20% by weight.

The composition for oral administration includes solid or liquid dosage forms, specifically tablets (including sugar-coated tablets, film-coated tablets), granules, powders, capsules (including soft capsules), syrup. Agents, emulsions or suspensions. Such a composition is produced by a method known per se and contains a carrier usually used in the field of formulation. Such carriers are physiologically acceptable carriers, and various conventional organic or inorganic carrier substances are used as formulation materials. Excipients, lubricants, binders, disintegrants, etc. in solid formulations; in liquid formulations It is used as a suspending agent. Also, if necessary,
Formulation additives such as preservatives, antioxidants, coloring agents and sweeteners can be appropriately selected and used in appropriate amounts.

Suitable examples of excipients include lactose,
White sugar, D-mannitol, starch, crystalline cellulose,
Light silicic anhydride and the like can be mentioned. Preferred examples of the lubricant include, for example, magnesium stearate, calcium stearate, talc, colloidal silica and the like. Preferable examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like. Preferable examples of the disintegrant include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, sodium carboxymethyl starch and the like.

Preferable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and glyceryl monostearate; Examples thereof include hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose. Preferable examples of the preservative include, for example, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferable examples of the antioxidant include, for example, sulfite, ascorbic acid and the like.

Examples of the composition for parenteral administration include injections and suppositories. The injections are subcutaneous injections,
It includes dosage forms such as intradermal injection and intramuscular injection. Such an injection is prepared as an aqueous solution by a method known per se, that is, by dissolving, suspending or emulsifying helioxanthin or a lactone cleaved product thereof or a salt thereof in a sterile aqueous liquid or oily liquid usually used for injections. Examples of the aqueous solution for injection include physiological saline and isotonic solution. If necessary, a suitable suspending agent such as sodium carboxymethyl cellulose, a nonionic surfactant and the like may be used in appropriate amounts together. Examples of the oily liquid include sesame oil and soybean oil, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule.

The dose for a particular patient depends on age, weight,
It may be determined according to general health conditions, sex, diet, administration time, administration method, excretion rate, drug combination, the degree of the medical condition of the patient being treated at the time, and other factors. The dose of the agent for enhancing the action of a cell differentiation inducing factor of the present invention is appropriately selected depending on the type and symptom of the above-mentioned disease.
g) In the case of oral administration per day, the amount of helioxanthin or its lactone cleavage product or its salt is about 0.
The dose may be 1 to 500 mg, preferably about 1 to 50 mg, more preferably about 3 to 50 mg. The dose for one dose is determined in consideration of such a daily dose and a dosage form. The number of administrations is not particularly limited, but is preferably 1 to 5 times / day, more preferably 1 to 3 times / day. Helioxanthin is known as a component of crude drugs as described above, and has low toxicity.

The agent for enhancing the action of a cell differentiation inducer of the present invention is
Since it has a strong osteogenesis promoting activity, it can be mixed with a carrier for bone reconstruction as an osteogenesis promoting agent for bone repair or bone transplantation. For example, the compound of the present invention can be used by adhering to or containing artificial bone made of metal, ceramic, or polymer. The artificial bone preferably has a porous surface so that the agent for enhancing the action of a cell differentiation inducing factor of the present invention can be released in a living tissue when the artificial bone is transplanted into a bone defect portion. The compounds of the present invention are suitable dispersants,
Disperse it in a binder, diluent, etc. (eg collagen, physiological saline, citric acid solution, acetic acid solution, hydroxyapatite, fibrin or a mixture thereof), apply or impregnate it on artificial bone, and dry. It can be attached or contained. Such artificial bone is transplanted to the bone defect portion and firmly fixed to the defect portion. The artificial bone immobilizing agent contains helioxanthin, which is an active ingredient, as a physiologically acceptable dispersion medium, a binder, a diluent, and other ingredients effective for bone regeneration (such as calcium) when used as a medicine. It can be prepared by mixing.
The artificial bone fixative can also be used so as to fill the space between the artificial bone to be transplanted into the bone defect portion of the host and the bone defect portion without adhering or containing the artificial bone fixative.
The parenteral composition described here can also be used by adhering or containing a protein that promotes bone formation such as BMP family.

[0025]

[Examples] The following will specifically describe the effects and embodiments of the cell differentiation inducer action enhancer of the present invention by showing Experimental Examples and Examples. However, these are merely examples, and the present invention is not limited. It is not limited.

Experimental Example 1 Alkaline phosphatase (A
LP) Induction of mouse osteoblastic cell line MC3T3-E1 with 10% fetal calf serum (FC
Α: Minimum essential medium (MEM: min) containing S: fetal calf serum
Seeding a 96-well plate in the imun essential medium (8
000 / well), 2 days later BMP-4 / 7 heterodimer (Japanese Patent Application 6-111)
(Described in No. 255) in a medium with or without 3 ng / ml Table 1
The sample diluted to the concentration shown in (1) was added to the cells grown on one side (confluent), and the culture was continued for 72 hours. After washing the plate once with saline, add the substrate solution at room temperature.
Incubated for 15 minutes. The reaction was stopped by adding 0.05 N sodium hydroxide, and the absorbance at 405 nm was measured.
As a result, as shown in Table 1, helioxanthin showed BMP
It was proved that the activity, that is, the induction of ALP production by BMP was enhanced, and that it had excellent ALP production-inducing activity alone with or without BMP.

[0027]

[Table 1]

Experimental Example 2 Elongation of neurites in rat adrenal medulla-derived cell line PC12 cells suspended in Dulbecco MEM supplemented with 10% FCS (rat adrenal medulla pheochromocytoma; 2000 / wel)
l) was mixed with a sample containing various concentrations of NGF and helioxanthin shown in FIG. 1 and seeded on a 96-well plate,
Cultured for 3 days. After removing the culture solution, hematoxylin / eosin staining was performed using a commercially available kit [Diffquick R, International Reagents Co., Ltd., Kobe City]. The results of evaluation of neurite outgrowth by microscopic observation are shown in FIG. NGF 10ng / m
By adding 1 μM or 10 μM of helioxanthin in the presence of l, significant elongation of neurites of the sample cells was observed. That is, 1 μM or 10 μM in the presence of NGF 10 ng / ml
The addition of helioxanthin showed an effect equal to or higher than the effect of 100 ng / ml treatment with NGF alone, and the activity of helioxanthin to enhance nerve growth factor action was observed.

Experimental Example 3 Calcification promoting action of mouse osteoblast cell line by helioxanthin Mouse osteoblast cell line MC3T3-E1 was seeded on a 24-well plate at 10 ⁴ / well, and from the next day in the presence of 3 ng / ml BMP or 10% FCS, 10 mM β-gl containing various concentrations of helioxanthin in the absence
It was cultured for 10 days in α-MEM medium containing ycerophosphate and 50 μg / ml ascorbic acid. After washing the cells once with PBS, 6N
0.2 ml of hydrochloric acid was added. After 5 minutes, this solution was collected and the calcium content was measured using a colorimetric assay kit (calcium E-test Wako, Wako Pure Chemical Industries, Ltd.) (n = 3). As a result, FIG.
As shown in Fig. 5, no clear calcification was observed by BMP treatment alone, whereas co-treatment with 10 or 1 μM helioxanthin significantly promoted calcification, and helioxanthin revealed a differentiation-inducing action by BMP. It was found to increase to. Furthermore, treatment with 10 μM helioxanthin alone significantly promoted calcification as compared with the untreated group, which is considered to be due to the enhancement of the action of endogenous BMP.

Experimental Example 4 Effect of Helioxanthin on Differentiation of Rat Bone Marrow Stromal Cells into Osteoblasts Stromal cells obtained from the femurs of 5-week-old male SD rats were seeded at 10 ⁴ / well on a 24-well plate. However, from the next day, 15% FCS containing various concentrations of helioxanthin, 10 mM β-glycerophosphate, 5
Α-M containing 0 μg / ml ascorbic acid, 10 ^-7 M dexamethasone
The cells were cultured in EM medium for 13 days. Cells were plated on days 4, 7, 11, and 13 of culture.
After washing once with BS, the alkaline phosphatase activity was measured by directly adding the substrate solution (see Experimental Example 1) to the cells (n = 3). In addition, 0.2 ml of 6N hydrochloric acid was added, the solution was recovered, and the calcium content was determined by a colorimetric assay kit (calcium E-
Test Wako, Wako Pure Chemical Industries, Ltd. (n = 3). As a result, as shown in FIG. 3, helioxanthin increased alkaline phosphatase activity in a dose-dependent manner, and they became more prominent as the culture period became longer. Further, as shown in FIG. 4, it was revealed that calcification was rapidly promoted from the 11th day of culture in the helioxanthin-treated group. From the above results, it is shown that helioxanthin has an action of acting on osteoblast precursor cells in vivo to promote their maturation and differentiation. This action of helioxanthin is considered to be the result of enhancing stimulation by low concentration of endogenous BMP.

Example 1 The ingredients (1) to (6) shown below were mixed to prepare about 1,000 tablets each having a diameter of 6.5 mm and containing 5 mg of helioxanthin per tablet in a dosage form forming machine. This is coated with the components (7) to (9) shown below to obtain a film-coated tablet having a diameter of 6.6 mm. (1) Helioxanthin 5g (2) Lactose 82.5g (3) Hydroxypropylcellulose 2.8g (4) Magnesium stearate 0.4g (5) Hydroxypropylmethylcellulose 2910 2.994g (6) Corn starch 19.3g (7) Macrogol 6000 0.6g (8) Titanium oxide 0.4g (9) Ferric sesquioxide 0.006g

Example 2 The following components (1), (3), (4), (5), (6), (7) and (8) were suspended or dissolved in purified water and Fine particles can be produced by coating the core particles of (2). The ingredients (9) to (11) shown below are coated on the fine granules to form coating fine granules, which are mixed with the following ingredient (12) to prepare helioxanthin fine granules 1%, about 500 g. obtain. This is divided into 500 mg each. (1) Helioxanthin 5g (2) Lactose / crystalline cellulose (granules) 330g (3) D-mannitol 29g (4) Low-substituted hydroxypropylcellulose 20g (5) Talc 25g (6) Hydroxypropylcellulose 50g (7) Aspartame 3g (8) Dipotassium glycyrrhizinate 3g (9) Hydroxypropylmethylcellulose 2910 30g (10) Titanium oxide 3.5g (11) Yellow ferric oxide 0.5g (12) Light anhydrous silicic acid 1g

[0033]

Industrial Applicability The agent for enhancing the action of a cell differentiation inducer of the present invention has, for example, a strong BMP action-enhancing activity and bone formation promoting activity, and acts on bone tissue to increase bone mass and bone strength. Therefore, this agent is useful for the treatment and prevention of various bone diseases such as osteoporosis, bone fracture healing promotion and bone reconstruction promotion. In addition, this drug has a neurotrophic factor action-enhancing activity,
It is useful for treating and preventing various neurological diseases such as Alzheimer's dementia and general senile dementia, motor neuron disorder (amyotrophic lateral sclerosis, etc.), and diabetic peripheral neuropathy.

[Brief description of the drawings]

FIG. 1 shows the neurite outgrowth ability of a rat adrenal medulla-derived cell line by helioxanthin.

FIG. 2 shows the calcification promoting action of mouse osteoblastic cell lines by helioxanthin. The average value and standard deviation value of 3 holes per group are shown. * Indicates that a statistically significant difference from the group treated with BMP alone was observed (p <0.05, t-test). # Indicates that a statistically significant difference from the untreated control group was observed (p <0.05, t-test).

FIG. 3 shows an action of promoting differentiation of rat bone marrow stromal cells into osteoblasts by helioxanthin. The average value and standard deviation value of 3 holes per group are shown. **, * Indicates that a statistically significant difference from the untreated control group was observed (**; p <0.01, *; p <0.05, t-test).

FIG. 4 shows an action of promoting differentiation of rat bone marrow stromal cells into osteoblasts by helioxanthin. The average value and standard deviation value of 3 holes per group are shown. **, * Indicates that a statistically significant difference from the untreated control group was observed (**; p <0.01, *; p <0.05, t-test).

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Claims (9)

[Claims] 1. A cell differentiation inducing factor action enhancer comprising helioxanthin, a lactone cleaved product thereof, or a salt thereof. 2. The agent for enhancing the action of a cell differentiation inducer according to claim 1, wherein the cell differentiation inducer is an osteogenic factor. 3. The agent for enhancing the action of a cell differentiation inducer according to claim 1, wherein the cell differentiation inducer is a neurotrophic factor. 4. The neurotrophic factor is neurotrophic
The agent for enhancing the action of a cell differentiation inducing factor according to claim 3, which is a factor family. 5. A composition for the treatment / prevention of bone diseases, which comprises helioxanthin, a lactone cleaved product thereof, or a salt thereof. 6. A therapeutic / preventive composition for a neurodegenerative disease, which comprises helioxanthin, or a cleaved lactone thereof or a salt thereof. 7. A composition comprising helioxanthin, a lactone cleavage product thereof or a salt thereof, and a cell differentiation inducing factor. 8. A method for enhancing the action of a cell differentiation inducing factor, which comprises using helioxanthin, a lactone cleaved product thereof, or a salt thereof. 9. An osteogenesis promoter comprising helioxanthin, a lactone cleavage product thereof, or a salt thereof.