JP2002027998A - Method for measuring activity for regulating degeneration of nerve cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To search a medicine for treating central nervous system diseases. SOLUTION: It was found that a nerve cell degeneration was caused by a fibroblast growth factor and/or an eicosanoid, and a method for measuring the regulation activity of a compound against the nerve cell degeneration caused by the fibroblast growth factor and/or the eicosanoid is also found.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring neuronal degeneration regulating activity in nerve cells. More specifically, a method for measuring the activity of enhancing or inhibiting the degeneration of nerve cells by fibroblast growth factor and / or eicosanoid (for example, prostaglandin), a test kit for the measurement method, and a method using the measurement method A compound other than a prostaglandin D ₂ receptor inhibitor and a peroxisome proliferator-activated receptor γ inhibitor obtained by
The present invention relates to the use of the compound as a therapeutic agent for Alzheimer's disease or stroke.

[0002]

BACKGROUND OF THE INVENTION Eicosanoids are known for Alzheimer's disease (Iwamoto et al., (1989) J. Neurol. 236,80-84) or stroke (Hsu et al., (1989) Ann. NY Acad. Sci. 559, 282-
295) and so on. JP 62
To -198,616 (1987 September 2 published), the prostaglandin D _2, which is one of the eicosanoids, have a nerve cell protective action is disclosed. Also, 15-deoxy-, a metabolite of prostaglandin D ₂
Δ ^12,14 -prostaglandin J ₂ has been reported to induce apoptosis in proliferating cells (Chatt
opadhyay et al., (2000) J. Neurosci. Res. 61, 67-.
74). Furthermore, it is clear that 15-deoxy-Δ ^12,14 -prostaglandin J ₂ induces apoptosis in proliferating cells via its receptor, peroxisome proliferator-activated receptor γ. (But
ler et al., (2000) Cell Growth Differ. 11, 49-6.
1). On the other hand, for nerve cells that are non-proliferating cells,
15-deoxy-Δ ^12,14 -prostaglandin J ₂ has been found to have a process extension effect (Sato et al.,
(1999) Biochem. Biopys Res Commun. 258, 50-5.
3). It has been known that administration of thromboxane A ₂ , another eicosanoid other than prostaglandin D ₂ , to the unilateral striatum of mice disrupts the balance of striatal function and induces rotational behavior (WO00 / 30683, June 2000
2 days). However, it has not yet been reported that metabolites of prostaglandin D ₂ including prostaglandin D ₂ and 15-deoxy-Δ ^12,14 -prostaglandin J ₂ induce neuronal degeneration in nerve cells. Not even.

[0003] Fibroblast growth factor is one of cell growth factors and is known as a growth factor for fibroblasts. In addition, it has been reported that it has a neuroprotective effect on nerve cells (WO99 / 66959 (December 1999).
Released on March 29)). On the other hand, it has also been reported that mesangial cells, which are proliferating cells, induce neuronal degeneration (Floege et al., (1998) J. Am. Soc. Nephro
l. 9, 792-801). It has been reported that growth factors other than fibroblast growth factors, ciliary neurotrophic factor, leukemia inhibitory factor, and interleukin-6 induce neuronal death via membrane protein gp130 (Kessler et al.). ,
(1993) Neuron 11, 1123-1132). However, it has not yet been reported or suggested that fibroblast growth factor, which transmits information into cells through a receptor different from gp130, induces neuronal degeneration in nerve cells.

[0004]

Under the above circumstances, a method for searching for a therapeutic agent for a central nervous system disease based on the activity of regulating neuronal degeneration and a useful therapeutic agent for a central nervous system disease obtained by the method are desired. Was.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to elucidate the physiological functions of fibroblast growth factors and eicosanoids (for example, prostaglandins). And it was found that eicosanoids induce neuronal degeneration in nerve cells, and completed the present invention. That is, the present invention provides a novel method for measuring neuronal degeneration-regulating activity in nerve cells, a method for measuring an activity for enhancing or inhibiting fibroblast growth factor or eicosanoid (for example, prostaglandin, etc.) on degeneration of nerve cells, Test kit for measuring method, compound other than prostaglandin D ₂ receptor inhibitor and peroxisome proliferator-activated receptor γ inhibitor obtained by using the measuring method, containing the compound as an active ingredient Provided is a therapeutic agent for stroke, Alzheimer's disease or cardiomyopathy.

[0006] Specifically, the present invention includes the following inventions. (1) A method for measuring the regulatory activity of a compound on neuronal degeneration caused by fibroblast growth factor and / or eicosanoid. (2) The method according to the above (1), comprising the following steps.
(A) contacting a test compound with a nerve cell;
(B) contacting the neuron with fibroblast growth factor and / or eicosanoid; (c) above (b)
Measuring the modulatory activity of the test compound on neuronal degeneration caused by the above. (3) contacting a test compound with a nerve cell and then contacting fibroblast growth factor and / or eicosanoid, and determining whether the test compound has a regulatory activity on neuronal degeneration caused by fibroblast growth factor and / or eicosanoid The method according to any one of (1) and (2) above, wherein (4) The method according to (3) above, wherein the measurement index of neuronal degeneration is a morphological change of a cell. (5) The method according to (3) above, wherein the measurement index of neuronal degeneration is a decrease in mitochondrial function in cells. (6) The method according to (3) above, wherein the measurement index of neuronal degeneration is an increase in cytoplasmic component leakage in the cell. (7) The method according to (3), wherein the measurement index of neuronal degeneration is a decrease in uptake of nucleic acid in cells. (8) The method according to (3) above, wherein the measurement index of neuronal degeneration is an increase in nuclear accumulation of a fluorescent substance in cells. (9) The method according to (3) above, wherein the measurement index for neuronal degeneration is an increase in chromatin aggregation in the cells. (10) The measurement index of neuronal degeneration is DN in cells
The method according to (3) above, which is an increase in A fragmentation. (11) The fibroblast growth factor is a fibroblast growth factor-
2. The method according to any one of (1) to (10) above, wherein (12) The method according to any one of (1) to (10) above, wherein the eicosanoid is a prostaglandin. (13) Prostaglandin is Prostaglandin D
_2. The method according to (12) above, which is ₂ or a metabolite thereof. (14) Prostaglandin J
_2. The method according to (12) above, which is ₂ or a metabolite thereof. (15) The method according to the above (12), wherein the prostaglandin is Δ ¹² -prostaglandin J ₂ or a metabolite thereof. (16) Prostaglandin is 15-deoxy-Δ
^{12, 14} - prostaglandin J ₂ or its metabolites, The method according to (12). (17) nerve cells and fibroblast growth factor and / or
Alternatively, a kit for measuring neuronal degeneration regulating activity containing eicosanoid. (18) A compound, a prodrug or a pharmaceutically acceptable salt thereof, or a hydrate thereof obtained by using the measuring method according to any one of the above (1) to (16). (19) The compound according to (18) above, wherein the compound is other than a prostaglandin D ₂ receptor inhibitor, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a hydrate thereof. (20) The compound according to (18) above, wherein the compound is other than a peroxisome proliferator-activated receptor γ activity modifier.
A prodrug thereof, a pharmaceutically acceptable salt thereof, or a hydrate thereof. (21) A therapeutic agent for central nervous system diseases, comprising the compound according to (19) and / or (20), a prodrug thereof, a pharmaceutically acceptable salt thereof, or a hydrate thereof. (22) A therapeutic agent for stroke or Alzheimer's disease, comprising the compound according to (19) and / or (20), a prodrug thereof, a pharmaceutically acceptable salt thereof, or a hydrate thereof. Equation (23):

Embedded image (Wherein R is each independently an alkyl), a therapeutic agent for a central nervous system disease, which comprises, as an active ingredient, a compound represented by the following formula: or a prodrug thereof, a pharmaceutically acceptable salt thereof, or a hydrate thereof.

Hereinafter, terms used in the present specification will be described. "Eicosanoid" means a physiologically active substance derived from arachidonic acid. For example, thromboxane (e.g., such as thromboxane A _2), prostaglandins (e.g., prostaglandin D _2, prostaglandin J _2, delta ¹² - prostaglandin J _2, 15-deoxy - [delta ^{12, 14} - Prostaglandin J ₂ , Prostaglandin E ₂ , Prostaglandin I ₂ , Prostaglandin F
₂ alpha, prostaglandins such as prostaglandin A _2), leukotrienes (e.g., leukotriene A _4, leukotriene B _4, leukotriene C _4, leukotriene D _4, leukotriene E _4), such as hydroxy eicosatetraenoic acid. In particular, prostaglandins are preferred. "Prostaglandin" means a physiologically active substance produced by arachidonic acid from cyclooxygenase. For example, prostaglandin D ₂ , prostaglandin J ₂ , Δ ¹² -prostaglandin J ₂ , 15-deoxy-
Δ ^12,14 -prostaglandin J ₂ , prostaglandin E ₂ , prostaglandin I ₂ , prostaglandin F ₂
α, prostaglandin A _{2 and the} like. In particular,
Prostaglandin D ₂ , Prostaglandin J ₂ , Δ ¹²
―Prostaglandin J ₂ , 15-deoxy-Δ ^12,14 ―
Prostaglandin J ₂ is preferred. “Prostaglandin D ₂ ” means a prostaglandin produced by prostaglandin D ₂ synthase. “Fibroblast growth factor” was first isolated and purified by Gospodarowicz (Gospodarowicz, (1975) J. Biol. Chem. 2
50, 2515-2520), which means a group of proteins having fibroblast growth promoting activity. For example, fibroblast growth factor-2 and the like can be mentioned. “Peroxisome proliferator-activated receptor γ activity modifier” means an activator or inhibitor of peroxisome proliferator-activated receptor γ. "Modulatory activity" means enhancing activity or inhibitory activity, preferably inhibitory activity.

[0008] The measuring method of the present invention also includes a measuring method using the following phenomena resulting from neuronal degeneration as indices.
"Morphological changes" refers to morphological changes when nerve cells are observed with a light microscope or an electron microscope, for example, neurite degeneration, cell body atrophy, cell membrane foaming, Abnormality, nuclear fragmentation, etc. “Mitochondrial function” means the redox ability of mitochondria. “Leakage of cytoplasmic components” means leakage of cytoplasmic components including lactate dehydrogenase out of cells due to some cell membrane damage. "Nucleic acid uptake" means the uptake of nucleic acids such as thymidine into cells. “Nuclear accumulation of fluorescent substance” means accumulation of a fluorescent substance such as propidium iodide in a cell nucleus due to some cell membrane damage. "DN
"A fragmentation" refers to the cleavage of DNA in cell nuclei.

More specifically, experiments were conducted on the effects of fibroblast growth factor and eicosanoids (eg, prostaglandin) on neuronal degeneration (eg, neuronal cell death), and a compound having an inhibitory effect was found. Adjustment of nerve cells can be performed as follows.
First, the cerebral cortex or hippocampus is removed and suspended in an isotonic solution, and then protease alone is added or D
Add together with NA-degrading enzyme, and further add
The cells are cultured at (preferably 25 ° C to 38 ° C) for 5 minutes to 20 minutes (preferably 10 minutes to 15 minutes) to disperse the nerve cells. Next, the nerve cells thus obtained are suspended in a medium, and the nerve cells suspended in the medium are spread on a culture plate coated to prevent adsorption.

As the isotonic solution, for example, salts such as sodium chloride, potassium chloride, sodium phosphate and potassium phosphate, and saccharides such as glucose and sucrose may be used in combination. As a protease, for example,
Trypsin, chymotrypsin, collagenase, thrombin, plasmin, elastase, papain (0.4-
4 mg / ml), and preferably trypsin. D
As the NA-decomposing enzyme, for example, deoxyribonucleases I and II, preferably, deoxyribonuclease I are used. As the medium, for example, Leivbovitz's L
-15 medium, Eagle's Basic medium, Eagle's MEM medium, F10 medium, Dulbecco's MEM medium, RPM-1640 medium, F12 medium, Is
Cove's MDM medium, McCoy's5a and the like can be used. As the coating agent for the plate, poly-L-lysin, collagen, polyethyleneimine, polyornithine and the like can be used.

A method for measuring the regulatory activity of a test compound on neuronal degeneration by fibroblast growth factor may be performed as follows. The above-prepared nerve cells were treated with (i) fibroblast growth factor-2 (0.1 ng / ml to 10 ng / ml).
ml, preferably 0.1-3 ng / ml) or (ii) fibroblast growth factor-2 (0.1 ng / ml).
〜１０10 ng / ml, preferably 0.3 ng / ml〜3 n
g / ml) and the test compound (0.01 ng / ml to 1)
ng / ml, preferably 0.03 ng / ml to 0.3
ng / ml). After 24 hours to 72 hours (preferably 36 hours to 60 hours) after the addition, the neuronal degeneration of the groups (i) and (ii) is measured using the phenomenon resulting from the neuronal degeneration as an index.

In addition, a method for measuring the modulatory activity of a test compound on neuronal degeneration caused by eicosanoids (eg, prostaglandins) may be performed as follows. (I) Prostaglandin D
₂ (1 μM to 30 μM, preferably 3 μM to 10 μM
M), prostaglandin J ₂ , Δ ¹² -prostaglandin J ₂ or 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (0.3 μM to 10 μM, preferably 0.6 μM to
3 μM) alone or (ii) prostaglandin D ₂
(1 μM to 30 μM, preferably 10 μM), prostaglandin J ₂ , Δ ¹² -prostaglandin J ₂ or 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (0.3
μM to 10 μM, preferably 0.6 μM to 3 μM) and the test compound (0.1 μM to 30 μM, preferably 0.1 μM to 0.3 μM).
3 μM to 10 μM). 4 hours to 24 hours after the addition (preferably 8 hours to 18 hours after the addition), the neuronal degeneration of the groups (i) and (ii) is measured using the phenomenon resulting from the neuronal degeneration as an index. The test compound may be administered simultaneously with the fibroblast growth factor and / or eicosanoid, or may be administered separately. In particular, it is preferable to administer the test compound before administering the fibroblast growth factor and / or eicosanoid.

Whether or not a test compound has a regulatory activity on neuronal degeneration caused by fibroblast growth factor and / or eicosanoid may be determined by using a phenomenon resulting from the neuronal degeneration as an index. Hereinafter, a measurement method using “decrease in mitochondrial function” as an index will be described.
MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) is added to the cell culture solution, and the mixture is added at 20 ° C to 40 ° C (preferably 25 ° C to 38 ° C). For 30 minutes to 180 minutes (preferably 60 minutes to 120 minutes), and the produced formazan crystals are dissolved in propanol and measured at a specific wavelength of an automatic microplate reader. That is, the mitochondrial respiratory activity of the cells is measured, and the measured values of the groups (i) and (ii) are compared to determine whether the test compound has a neuronal degeneration-enhancing activity or an inhibitory activity. do it. The measurement method of the present invention uses not only a case where a compound whose regulatory activity on neuronal degeneration is unknown but also a compound whose regulatory activity on neuronal degeneration has been confirmed in another experimental system as a test compound. It is also included. That is, the case where the activity of regulating neuronal degeneration is reconfirmed is also included. The present invention also encompasses a kit used for the above-described measurement method, that is, a kit for measuring neuronal degeneration regulating activity containing nerve cells and fibroblast growth factor and / or eicosanoid. The compound selected by the above-mentioned measurement method, in particular, a compound having an activity of inhibiting neuronal degeneration is useful as a neuroprotective agent (for example, an inhibitor of neuronal degeneration, an inhibitor of neuronal death), and furthermore, a central nervous system disease It is useful as a therapeutic agent (particularly, a therapeutic agent for stroke or Alzheimer's disease). That is, it exerts a neuronal degeneration inhibitory activity on neuronal degeneration that can be caused by a central nervous system disease, and can treat or prevent a central nervous system disease. Examples of central nervous system diseases include Alzheimer's disease, Parkinson's disease, schizophrenia, cerebellar degenerative agents, Wilson's disease, Down's syndrome, retinitis pigmentosa, Lewy body disease, cerebral ischemia, muscular atrophy Lateral sclerosis, prion-related disease, Creutzfeldt-Jakob disease, Kru's disease, multiple sclerosis, hereditary ataxia, Scheidlegger syndrome, progressive supranuclear syndrome, Huntington's chorea, spinal muscular atrophy, Reye's syndrome, status epilepticus, progressive multifocal white matter brain, viral encephalitis, normal pressure hydrocephalus, subacute sclerosing panencephalitis, degeneration after cerebrospinal injury,
These include frontal lobe dementia, gray myelitis, glaucoma, autonomic neuropathy, Guillain-Barre disease, diabetic neuropathy, porphinosis, autoimmune disease, vasculitis and the like. Eicosanoids have been identified in Alzheimer's disease (Iwamoto et al., (1989) J. Neurol. 236, 8
0-84) or stroke (Hsu et al., (1989) Ann. NYAcad. S)
ci. 559, 282-295), the compounds selected by the measurement method of the present invention are particularly useful for preventing and / or treating stroke or Alzheimer's disease. Useful as Examples of the compound include an organic compound having a molecular weight of 15 to 1,000, and the constituent atoms include hydrogen, lithium, boron, carbon, nitrogen, oxygen, fluorine, sodium, magnesium, aluminum, sulfur, chlorine, potassium, calcium, Iron, barium, bromine, iodine and the like. In particular,
Compounds having a group represented by -C (= O)-or -NH- are preferred. Further, a compound having an unsaturated bond is preferable. Further, as the compound, prostaglandin D ₂
Compounds other than the receptor inhibitor and compounds other than the peroxisome proliferator-activated receptor γ activity modifier are preferred. Compounds having an inhibitory activity on fibroblast growth factor and / or eicosanoid-induced neuronal degeneration include compounds represented by the formula:

Embedded image (Wherein R is each independently an alkyl), or a pharmaceutically acceptable salt thereof, or a hydrate thereof. "Alkyl" means a straight-chain or branched alkyl group having 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-
Means butyl. By prodrug of a compound according to the present invention is meant a derivative of a compound according to the present invention having a group that can be chemically or metabolically degraded, wherein the pharmaceutically active compound of the present invention is in vivo under solvolysis or under physiological conditions. Is a compound that becomes a compound of Methods for selecting and producing suitable prodrug derivatives are described, for example, in Design of Prodrugs,
Elsevier, Amsterdam 1985. When the compound according to the present invention has a carboxyl group, an ester derivative produced by reacting the original acidic compound with an appropriate alcohol, or an ester derivative produced by reacting the original acidic compound with an appropriate amine Prodrugs such as the amide derivatives described above are exemplified. Particularly preferred esters as prodrugs include methyl ester, ethyl ester, n-propyl ester, isopropyl ester, n
-Butyl ester, isobutyl ester, tert-butyl ester, morpholinoethyl ester, N, N-diethylglycolamide ester and the like. When the compound according to the present invention has a hydroxyl group, examples thereof include prodrugs such as acyloxy derivatives produced by reacting a compound having a hydroxyl group with a suitable acyl halide or a suitable acid anhydride. Particularly preferred acyloxy as _{prodrugs, -OCOC 2 H 5, -OCO (} t-Bu), - O
COC ₁₅ H ₃₁ , -OCO (m-COONa-Ph),-
OCOCH ₂ CH ₂ COONa, —OCOCH (NH ₂ )
CH ₃ , —OCOCH ₂ N (CH ₃ ) _{2 and the} like. When the compound according to the present invention has an amino group, a prodrug such as an amide derivative produced by reacting the compound having an amino group with a suitable acid halide or a suitable mixed acid anhydride is exemplified. . Amides particularly preferred as prodrugs include -NHCO
_{(CH 2) 20 CH 3,} -NHCOCH (NH 2) CH 3 and the like. In particular, since the present invention relates to a prophylactic and / or therapeutic agent for a central nervous system disease, it is preferable to use a prodrug that enhances translocation to the brain (center). In particular, it is important to penetrate the blood-brain barrier (BBB). Generally, in order to enhance the transferability to the center, a protective group having high lipophilicity is introduced.
Particularly, in the case of a low molecular weight compound, since a good correlation has been recognized between BBB permeability and lipophilicity (eg, LogP value, cLogP value), a protecting group to be introduced is selected with reference to those values. (Oldendorf (1974) Pro
c. Soc. Exp. Biol. Med. 147, 813-816). Also, Bodo
(1983) Pharmacol. Ther. 19, 337-386, a drug delivery system utilizing the Locking in phenomenon may be used. Therefore, even a parent compound having high hydrophobicity can be used as a prophylactic and / or therapeutic agent for central nervous system diseases by making such a prodrug. Invasive methods to promote brain (central) migration include intraventricular and intracerebroventricular administration, Osmoti
c Opening method, use of permeability enhancer, etc. (Development of Drugs, Drug Delivery Method, Vol. 13, 280-294 Hirokawa Shoten). In addition, the drug can be delivered to the brain by encapsulating the drug in liposomes prepared with brain-derived lipids (Naoi et al. (1980) Biochem. Int. 1,591).
1-596). Pharmaceutically acceptable salts of the compound of the present invention or a prodrug thereof include alkali metal salts (eg, lithium salt, sodium salt or potassium salt), alkaline earth metal salts (eg, calcium salt), and the like. Organic bases (e.g., tromethamine, trimethylamine, triethylamine, 2-aminobutane, t-butylamine, diisopropylethylamine, n-butylmethylamicyclohexylamine, dicyclohexylamine, N-isopropylcyclohexylamine, furfurylamine, benzylamine, methylbenzylamine, dibenzylamine) Benzylamine, N, N-dimethylbenzylamine, 2
-Chlorobenzylamine, 4-methoxybenzylamine, 1-naphthylenemethylamine, diphenylbenzylamine, triphenylamine, 1-naphthylamine, 1
-Aminoanthracene, 2-aminoanthracene, dehydroapiethylamine, N-methylmorpholine or pyridine) or an amino acid salt (for example, a lysine salt or an arginine salt). Hydrates are compounds of the present invention, prodrugs thereof,
Or hydrates of their pharmaceutically acceptable salts,
For example, monohydrate and dihydrate can be mentioned. The compounds according to the present invention include all stereoisomers (diastereomers, epimers, enantiomers and the like) or racemates. Further, when the compound of the present invention has a double bond, Z-form and E-form may exist,
The invention includes compounds having either configuration, or a mixture of both. The compounds can be administered orally or parenterally. In the case of oral administration,
The compound may be used in any of the usual pharmaceutical forms, for example, solid preparations such as tablets, powders, granules and capsules; liquid preparations; oily suspensions; and liquid preparations such as syrups and elixirs. Can be. In the case of parenteral administration, the above compound can be used as an aqueous or oily suspension injection and nasal drops. In the preparation thereof, conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents, preservatives, stabilizers and the like can be optionally used. The formulations of the present invention are prepared by combining (eg, mixing) a therapeutically effective amount of the above compound with a pharmaceutically acceptable carrier or diluent. Formulations of the above compounds are prepared in a known manner using well-known and readily available ingredients. In preparing pharmaceutical compositions of the compounds described above, the active component may be mixed with or diluted with a carrier, or placed in a carrier in the form of a capsule, sachet, paper, or other container. When the carrier acts as a diluent, the carrier is a solid, semisolid, or liquid material that acts as a vehicle, and may be tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions. Syrups, aerosols (solids in liquid medium), ointments and contain, for example, up to 10% of active compound. The compound of the present invention is preferably formulated before administration. Any suitable carrier known to those skilled in the art can be used for this formulation. In such formulations, the carrier is a solid, liquid, or mixture of a solid and a liquid. For example, for intravenous injection, 2 mg /
4% dextrose / 0.5% to a concentration of 0.5 ml
Dissolve in aqueous sodium citrate solution. Solid formulations include powders, tablets and capsules. A solid carrier is one or more substances that also serve as ingredients for flavoring, lubricants, dissolving agents, suspending agents, binders, tablet disintegrants, and capsules. Tablets for oral administration include calcium carbonate, sodium carbonate, lactose with disintegrants such as corn starch, alginic acid, and / or binders such as gelatin, acacia, and lubricants such as magnesium stearate, stearic acid, talc. And suitable excipients such as calcium phosphate. In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active ingredient is in the proper ratio,
It is mixed with a carrier having the necessary binding properties and is hardened to a desired shape and size. About 1 to 1 powder and tablet
It contains about 99% by weight of the active ingredient which is a novel compound according to the invention. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter.
Liquid preparations include suspensions, emulsions, syrups, and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent, or a mixture of both. The active ingredient can often be dissolved in a suitable organic solvent, for example, aqueous propylene glycol. Other compositions can be prepared by dispersing the finely divided active component in aqueous starch, sodium carboxymethyl cellulose solution, or a suitable oil. The dose of the above compound varies depending on the method of administration, the age, weight, condition and type of disease of the patient. However, in the case of oral administration, it is usually about 0.05 mg to 3000 mg, preferably about 0.5 mg / day for an adult. 1 mg to 1000 mg may be administered in divided doses, if necessary. In the case of parenteral administration, about 0.01 mg to 1000 mg, preferably about 0.05 mg / day for an adult.
mg to 500 mg are administered.

Hereinafter, the present invention will be described in more detail with reference to Examples.
Are described, but these limit the scope of the present invention.
is not. Compound (A) refers to herbimycin A
(C ₃₀H₄₂N_TwoO₉) And the compound (B) is (5Z)
-7-[(1R, 2R, 3S, 5S) -2- (4-phenylsulfani
Ru-benzoylamino) -6,6-dimethylbicyclo [3.
1.1] Hept-3-yl] -5-heptenoate sodium
means.

Example 1: By fibroblast growth factor-2
The cerebral cortex was removed from a Sprague-Dawley rat (embryonic day 19) according to the method described in Developmental Brain Research, 30 (1986) 47-56 for nerve cell death , and an isotonic solution (137 mM sodium chloride, 5.4 mM potassium chloride, 0.17 mM disodium phosphate, 0.22 mM potassium phosphate, 5.5 mM glucose, 59
mM sucrose; 25 ml each). Trypsin and deoxyribonuclease were added at 4 mg / ml and 0.4 mg / ml, respectively, and cultured at 37 ° C. for 12 minutes to disperse the nerve cells. In a Leivbovitz's L-15 medium (containing 5% calf serum + 5% horse serum), 1x10
^The cells were suspended at ⁶ cells / ml. 2.5 x 10 ⁵ cells / cm ^{2 on} culture plate coated with poly-L-lysin
Nerve cells were seeded at a density of. Nerve cells are 5% CO _{2 at} 37 ° C
The cells were cultured in an incubator and used for experiments on the second day.

[0016] The nerve cells are provided with (i) fibroblast growth factor-
2 (fibroblast growth factor-2 / culture solution = 100 ng / m
l) of 10 μl per 1 ml of medium, (ii) fibroblast growth factor-2 (fibroblast growth factor-2 / culture solution = 1)
00 ng / ml) per 10 ml of medium and 10 μl of compound (A) (compound (A) / dimethylsulfoxide = 1 μmol)
/ ml or 1 μmol / ml) was added simultaneously with 1 μl per 1 ml of medium. As a control, untreated (fibroblast growth factor-
2 was not added). 48 hours after the addition of fibroblast growth factor-2, 3- (4,
5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) was added to a concentration of 1 mg / ml, and the mixture was cultured at 37 ° C. for 1 hour. The produced formazan crystals were added to propanol (0.04 N hydrochloric acid)
MTT assay (mitochondrial activity) was performed using an automatic micropre-trider at a wavelength of 570 nm. The number of cases per group was 4, and a significant difference test was performed using Dunnett's test. The results obtained are shown in the attached FIG. ** means P <
0.01 indicates a significant difference. From FIG. 1, it was confirmed that the compound (A) exhibited an inhibitory effect on neuronal cell death caused by fibroblast growth factor-2. Fibroblast growth factor-2 caused about 50% neuronal death. When compound (A) was simultaneously applied, cell death was suppressed in a concentration-dependent manner. Its ED ₅₀ value was about 1 mM.

Example 2: Prostaglandin-induced nerves
Neuronal cells cultured by the method described in Example 1 for cell death were used in the experiment. Prostaglandin D ₂ (prostaglandin / ethanol = 10 μmol / ml), prostaglandin J ₂ , Δ ¹² -prostaglandin J ₂ or 15-deoxy-Δ ^12,14 -prostaglandin J ₂ (Prostaglandin / ethanol = 1 μmol / ml) in 1 ml of medium (without serum)
Was added to the mixture. As a control, untreated (without addition of prostaglandin) was similarly performed.
Prostaglandin D ₂ , Prostaglandin J ₂ , Δ ¹²
-Prostaglandin J ₂ or 15-deoxy-Δ ^12,14-
_2, 4, 6, 8 hours after the addition of prostaglandin J2,
MTT was added to the neurons to evaluate neuronal degeneration. The number of cases per group was 4, and a significant difference test was performed using Dunnett's test. The results obtained are shown in the attached FIG. ** indicates that there is a significant difference at P <0.01 compared to the untreated group. FIG.
From prostaglandin D ₂ , prostaglandin J ₂ , Δ ¹² -prostaglandin J ₂ or 15-deoxy
-Δ ^12,14 - it was confirmed neural cell death-inducing effect on prostaglandin J _2.

[0018] Example 3: 15-deoxy -Δ ^{12, 14} - Pros
Compound on neuronal death by Taguranjin J ₂
Action of (B) Neurons cultured by the method described in Example 1 were used for the experiment. Neuronal cells, (i) 15-deoxy -Δ ^{12, 14} - prostaglandin J ₂ (prostaglandin / ethanol = 1 [mu]
mol / ml) per 1 ml of medium (without serum)
(Ii) 15-deoxy -Δ ^{12, 14} - prostaglandin J ₂
(Prostaglandin / ethanol = 1 μmol / ml) and 1 μl of compound (B) (compound (B) / dimethylsulfoxide = 1 μmol / ml or 1 μl) per 1 ml of medium (without serum)
mol / ml) was added simultaneously to 1 ml of the medium (containing no serum). As a control, untreated (without addition of prostaglandin) was similarly performed. 12 hours after the addition, an MTT assay (mitochondrial activity) was performed. The number of cases per group was 4, and a significant difference test was performed using Dunnett's test. The results obtained are shown in the attached FIG. ** means P <
0.01 indicates a significant difference. Than 3, compound (B) is 15-deoxy - [delta ^{12, 14} - prostaglandin J ₂
It has been confirmed that the compound has an inhibitory effect on nerve cell death caused by neutrophil.

[0019]

EFFECTS OF THE INVENTION The present invention provides a method for inhibiting neuronal degeneration caused by fibroblast growth factor and / or eicosanoid of the present invention.
A compound having a neuronal degeneration-modulating activity can be found by a method for measuring the modulatory activity of a compound. Compounds having neuronal degeneration regulating activity, particularly compounds having neuronal degeneration inhibitory activity, can be expected as useful medicines for central nervous system diseases, particularly Alzheimer's disease and stroke.

[Brief description of the drawings]

FIG. 1 shows the inhibitory effect of compound (A) on neuronal cell death by fibroblast growth factor-2.

FIG. 2. Neuronal death by prostaglandin D ₂ or its metabolites.

[3] 15-deoxy -Δ ^{12, 14} - prostaglandin J ₂
Effect of compound (B) on neuronal cell death due to.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 C12N 15/00 A F term (Reference) 2G045 AA40 CB01 CB30 DA77 GC30 4B024 AA11 CA01 4B063 QA01 QA18 QQ02 QQ20 QQ42 QR69 QR72 QS28 QS36 QX01 4C084 AA17 NA14 ZA021 ZA022 ZA161 ZA162 4C086 AA01 AA02 BC33 MA01 MA01

Claims (23)

[Claims] 1. A method for measuring the regulatory activity of a compound on neuronal degeneration caused by fibroblast growth factor and / or eicosanoid. 2. The method of claim 1, comprising the following steps. (A) contacting a test compound with a nerve cell;
(B) a step of contacting a neuron with a fibroblast growth factor and / or eicosanoid; and (c) a step of measuring a regulatory activity of a test compound on neuronal degeneration caused by the above (b). 3. A test compound is brought into contact with a nerve cell and then brought into contact with fibroblast growth factor and / or eicosanoid, and the test compound has a modulating activity on neuronal degeneration caused by fibroblast growth factor and / or eicosanoid. 2. The method according to claim 1, further comprising:
Or the method of any one of 2. 4. The method according to claim 3, wherein the measurement index of neuronal degeneration is a morphological change of a cell. 5. The method according to claim 3, wherein the measurement index of neuronal degeneration is a decrease in mitochondrial function in a cell. 6. The method according to claim 3, wherein the measurement index of neuronal degeneration is an increase in cytoplasmic component leakage in the cell. 7. The method of claim 3, wherein the measure of neuronal degeneration is a decrease in nucleic acid uptake in the cell. 8. The method according to claim 3, wherein the measurement index of neuronal degeneration is an increase in nuclear accumulation of a fluorescent substance in a cell. 9. The method according to claim 3, wherein the measurement index of neuronal degeneration is an increase in chromatin aggregation in a cell. 10. The method according to claim 3, wherein the measurement index of neuronal degeneration is an increase in DNA fragmentation in a cell. 11. The method according to claim 1, wherein the fibroblast growth factor is fibroblast growth factor-2. 12. The method according to claim 1, wherein the eicosanoid is a prostaglandin. 13. prostaglandin is a prostaglandin D ₂ or its metabolites, The method of claim 12. 14. prostaglandin, prostaglandin J ₂ or its metabolites, The method of claim 12. 15. prostaglandin, delta ¹² - prostaglandin J ₂ or its metabolites claim 12
The method described in. 16. prostaglandin, 15-deoxy - [delta ^{12, 14} - prostaglandin J ₂ or its metabolites, The method of claim 12. 17. A kit for measuring a neuronal degeneration regulating activity comprising a fibroblast growth factor or eicosanoid and a nerve cell. 18. A compound, a prodrug thereof, a pharmaceutically acceptable salt thereof or a hydrate thereof obtained by using the measuring method according to any one of claims 1 to 16. 19. The compound according to claim 18, wherein the compound is a compound other than a prostaglandin D ₂ receptor inhibitor, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a hydrate thereof. 20. The compound according to claim 1, wherein the compound is a compound other than a peroxisome proliferator-activated receptor γ activity modifier.
8. The compound according to 8, a prodrug thereof or a pharmaceutically acceptable salt thereof or a hydrate thereof. 21. Claim 19 and / or 20
Or a prodrug thereof or a pharmaceutically acceptable salt thereof or a hydrate thereof. 22. The method according to claim 19, wherein the first and second conditions are different.
Or a prodrug thereof or a pharmaceutically acceptable salt thereof or a hydrate thereof, for treating stroke or Alzheimer's disease. 23. The formula: (Wherein R is each independently an alkyl), a therapeutic agent for a central nervous system disease, which comprises, as an active ingredient, a compound represented by the following formula: or a prodrug thereof, a pharmaceutically acceptable salt thereof, or a hydrate thereof.