JP2001199890A - Cell-protecting agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare a cell-protecting agent hardly causing the pressure decrease by administering an effective amount for suppressing apoptosis. SOLUTION: This cell-protecting agent contains PGK1, PGK2 or bicyclo-PGE2 as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cytoprotective agent.

[0002]

2. Description of the Related Art PGs (prostaglandins) are known to exhibit various physiological activities in vivo as autakoids. By the way, recently, the relationship between PGs and cell death (apoptosis) has attracted attention. For example,
Certain PGs have been shown to suppress apoptosis. Of these, the relationship between the PGE ₁ and apoptosis, JP-A 8-277222 discloses, J. Neu
rochem. , Vol. 72, No. 5, pp. 1907-1914,
Published in 1999. However, P
Since GE ₁ has a vasodilatory effect and may cause a decrease in blood pressure, there is a concern about a clinical situation that is difficult to use depending on the patient and the condition.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide PGs which are excellent in cytoprotective action (apoptosis inhibitory action) and which do not cause a decrease in blood pressure at an effective dose.

[0004]

The present inventors have SUMMARY OF THE INVENTION As a result of promoting further research in view of the above circumstances, has a specific PG compounds have excellent cell protective action such as PGK _1, and the effective dosage The present invention was completed by finding that the amount did not cause a decrease in blood pressure.

[0005] The present invention relates to PGK ₁ , PGK ₂ , bicyclo P
The present invention relates to a cytoprotective agent containing GE ₂ , a salt thereof, or a derivative thereof as an active ingredient. The details will be described below.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The active ingredient of the cytoprotective agent of the present invention is PGK ₁ , PGK ₂ , bicyclo PGE ₂ , a salt thereof, or a derivative thereof.

PGK ₁ , PGK ₂ or bicyclo PGE
₂ is a known compound. By the way, PGK ₁ is (13
E, 15S) -15-Hydroxy-9,11-dioxo-prost-13-en-1-oic acid, P
GK ₂ is (5Z, 13E, 15S) -15- hydroxy-9,11-dioxo - prostaglandin 5,13-dien-1-Oiku Acid, bicyclo PGE ₂ is 7- (4
-Butyloctahydro-2,5-dioxo-1H-inden-1-yl) -5-heptanoic acid. These compounds can be prepared according to a known method. Commercial products are also available (Syman Chemical, USA).

In the present invention, the salt may be an alkaline earth metal salt of the above compound, for example, a sodium salt, a potassium salt or the like, or an alkali metal salt, for example, a calcium salt or the like.
Salts with amino compounds, for example, tris (hydroxymethyl) aminomethane, N-methylglucamine and the like are exemplified. The salt can be prepared according to a known method. For example, PGK ₁ , P with free carboxylic acid
GK ₂ or bicyclo PGE ₂ and a hydroxide for forming a salt (eg, sodium hydroxide, potassium hydroxide,
These salts can be formed by coexisting calcium hydroxide and the like in an aqueous solution.

In the present invention, examples of the derivative include an ester form and an amide form of the above compound. Examples of the ester form include an alkyl ester, an alkyl ether alkyl ester, an alkyloxycarbonylalkyl ester, an alkylcarbonyloxyalkyl ester, and the like. The number of carbon atoms is about 1 to 20 as a whole substituent. Alkyl may be linear or branched.
Specifically, methyl, ethyl, n-propyl, iso-
Propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl, n-octyl, n
-Decyl and the like.

Examples of the amide include alkyl amides and dialkyl amides. The number of carbon atoms is about 1 to 20 as a whole substituent. The definition of alkyl is as described above.

The derivative can be prepared according to a known method. For example, P with a free carboxylic acid
It can be synthesized by esterification or amidation using GK ₁ , PGK ₂ or bicyclo PGE ₂ .

The compound of the present invention can be formulated according to a known method. For example, solid preparations and liquid preparations can be prepared. Solid preparations include powders, tablets, capsules and the like. Examples of the liquid preparation include an ethanol liquid preparation, a cyclodextrin clathrate, a liposome, and a fat emulsion (see JP-A-8-277222).

Since the compound of the present invention has an excellent apoptosis-suppressing action, it suppresses apoptosis of cells, especially brain cells, nerve cells or kidney cells, and is useful as a cytoprotective agent. Thus, these compounds are useful for neurological disorders,
Prevention and treatment of neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal canal stenosis, and renal diseases such as nephritis, renal failure, glomerulonephritis, nephrotic syndrome, etc. Useful for
In addition, since it inhibits cell damage due to NO (nitric oxide), it is also useful for the prevention and treatment of renal diseases in which radicals such as acute renal failure, drug-induced renal failure, and chronic renal failure are involved in apoptosis. . In addition, since these compounds show substantially no vasodilatory / hypertensive action at an effective dose, they are also useful for patients and disease states that may be adversely affected by lowering blood pressure.

The administration route of the cytoprotective agent of the present invention includes
It may be oral or parenteral. The dose is
It can be appropriately increased or decreased according to the sex, age, medical condition, weight, etc. of the patient. Usually, 0.1 to 10 per adult per day
About 00 μg is exemplified.

[0015]

EXAMPLES The present invention will be described in more detail with reference to the following Examples and Experimental Examples, which by no means limit the scope of the present invention.

Example 1 (Cyclodextrin inclusion) Compound of the present invention (PGK ₁ , PGK ₂ or bicyclo PGE)
₂ ) A solution prepared by heating and dissolving β-cyclodextrin (257 mg) in water (6 ml) was added to a solution prepared by dissolving 17 mg in ethanol (0.2 ml), and the mixture was mixed at 45 ° C., and then returned to room temperature to precipitate a precipitate. This was left overnight at 0 ° C., filtered, washed with a 50% aqueous ethanol solution, and sterilized and dried to obtain a cyclodextrin inclusion complex.

Example 2 (Liposome formation) After dissolving 60 mg of egg yolk phosphatidylcholine and 11 mg of oleylamine in 5 ml of chloroform, the compound of the present invention (PGK ₁ , PGK ₂ or bicyclo PGE ₂ )
A solution prepared by dissolving 30 μg in 100 μl of ethanol was added, the mixture was placed in an eggplant-shaped flask, and the solvent was distilled off with a rotary evaporator. 1 ml of a 0.1 M isotonic phosphate buffer (pH 5) was added thereto, and the mixture was shaken, sonicated (sonicated), and centrifuged. .

Example 3 (Ethanol solution) The compound of the present invention (PGK ₁ , PGK ₂ or bicyclo PGE)
₂ ) An ethanol solution was obtained by dissolving 500 μg in 1 ml of ethanol. When used, it is used after being diluted with a physiological saline solution or a glucose solution.

Example 4 (Emulsion of fat) In 30 g of purified soybean oil, 5.4 g of highly purified egg yolk phospholipid, and the compound of the present invention (PGK ₁ , PGK ₂ or bicycloPG)
E ₂ ) 1.5 mg and oleic acid 0.72 g were added,
It melt | dissolved by heating at 40-75 degreeC. 200 ml of distilled water
Was added, and 7.5 g of glycerin of the Japanese Pharmacopoeia was added. The total amount was made up to 300 ml with distilled water at 20 to 40 ° C., and the mixture was roughly emulsified with a homogenizer. The mixture was further subjected to high-pressure emulsion with a Menton-Gaulin type homogenizer to obtain a homogenized fine fat emulsion. The average particle size of this emulsion is 0.15 to 0.5.
4 μm, and did not contain particles of 1 μm or more.

Experimental Example 1 The inhibitory effect of the compound of the present invention on apoptosis induction by amyloid beta peptide was examined. 1) Culture of rat cerebral cortical neurons A cortical site was extracted from the cerebrum of a fetal rat of 17 or 18 days of age under ice-cooling, cut into pieces, and then subjected to a nerve cell dispersion (SUM).
The cells were dispersed using (ILON). Thereafter, the cells were seeded at a density of 1.5 × 10 ⁵ cells / cm ² in a culture flask coated with polyethyleneimine in advance, cultured for 4 days, and then subjected to the next test. The culture solution was Neuroba to which B27 supplement (1/50 volume), 2-mercaptoethanol (27.5 μM), L-glutamic acid (25 μM) and glutamine (0.5 mM) were added.
A sal medium (manufactured by Gibco) was used.

2) Induction of apoptosis by amyloid beta peptide and addition of the compound of the present invention Amyloid beta peptide 25-35 (Aβ _25-35 ) was dissolved in distilled water to a concentration of 1 mM, and the solution was dissolved at 37 ° C. for about 1 hour. week incubation were prepared Aged-Aβ _{25-3 5.} Induction of apoptosis in nerve cells was performed at 10 μM of Aged-Aβ
Was performed by replacing the above medium containing _{2 5-35} (L-glutamic acid is excluded). The compound of the present invention (P
After dissolving GK ₁ , PGK ₂ and bicyclo PGE ₂ ) in ethanol, Aged-Aβ _25-35 was added to the culture at the same time. Its final concentration was 1 μM. P as control
GE ₁ was used. Each group performed 3 cases.

3) Detection of apoptosis Twenty-four hours after the induction of apoptosis, the cells were washed with PBS (-) and fixed with a 1% glutaraldehyde solution (PBS solution) at room temperature for 30 minutes. Next, PBS (-)
After washing twice with 1 mM Hoechst 33342 solution (PBS
Solution) for about 2 minutes. After that, under a fluorescence microscope, morphology of nuclear chromatin is observed in any four visual fields, and the number of normal cells and the number of apoptosis-positive cells (cells showing chromatin fragmentation or aggregation) are counted.
The apoptosis induction suppression rate (%) was calculated. Table 1 shows the results
Shown in

Experimental Example 2 The blood pressure lowering effect of the compound of the present invention was examined. Normal rats (male, weighing about 300 g) were anesthetized with intraperitoneal administration of Nembutal, and then fixed in a dorsal position. Next, a cannula is inserted into the carotid artery to measure the blood pressure using a pressure transducer (Nihon Kohden).
And linked to a polygraph (Nihon Kohden) for simultaneous recording. After dissolving the compound of the present invention in ethanol, it was diluted with a saline (200-fold) and administered via the tail vein at a dose of 100 μg / kg body weight, and the mean body blood pressure (MBP) was measured. The blood pressure lowering degree was calculated from the measured values before and after the administration. The PGE ₁ was used as a control. Each group performed 3 cases. Table 1 shows the results.

[0024]

[Table 1]

It has been found that the compounds of the present invention (PGK ₁ , PGK ₂ , bicycloPGE ₂ ) can sufficiently suppress apoptosis at a dose that does not substantially lower blood pressure.

Experimental Example 3 Apoptosis was induced in kidney cells by NO (nitric oxide). NRK52E cells, a rat tubular epithelial cell line (mainly of distal origin), were cultured on a type I collagen-coated chamber slide for 24 hours. afterwards,
The medium was changed to a serum-free medium, and apoptosis was induced by adding a NO donor, S-nitroso-L-glutathione (GSNO; 1 mM). The compound of the present invention (PGK ₁ , bicyclo PGE ₂ ) was added to GSNO at a final concentration of 1 μm.
M was added. After 24 hours, cells were fixed with 1% glutaraldehyde and cells were stained with Hoechst 33342. Three visual fields per slide were arbitrarily selected, and the ratio of the number of cells in which nuclear fragmentation and aggregation occurred to the total number of cells in the visual field (400-fold magnification) was calculated. The number of cases was 5. Table 2 shows the results.

[0027]

[Table 2] The numerical values indicate the average value ± standard error. ^** indicates that there is a significant difference with the vehicle group at a risk factor of less than 1% when tested by the Dunnett method.

Compounds of the present invention (PGK ₁ , bicyclo PG
E ₂ ) was found to suppress apoptosis in renal cells, specifically, to suppress GSNO-induced apoptosis in tubular epithelial cell lines (NRK52E cells).

Experimental Example 4 The compound of the present invention (PGK ₁ , PGK ₂ or bicyclo-PGE)
₂ ) After dissolving in ethanol, dilute with saline (200
Fold) and normal rats (male, about 300 g in weight) were administered via the tail vein at a dose of 100 μg / kg body weight, but no lethal cases were observed.

[0030]

The compound of the present invention is useful as a cytoprotective agent (apoptosis inhibitor), particularly as a protective agent for brain cells, nerve cells and kidney cells. Specifically, neurological diseases, diseases associated with neurodegeneration, Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, spinal canal stenosis,
Alternatively, it is useful for prevention and treatment of renal diseases such as nephritis, renal failure, glomerulonephritis, nephrotic syndrome and the like. Also, N
Because it suppresses cell damage caused by O (nitric oxide),
It is also useful for prevention and treatment of renal diseases in which radicals such as acute renal failure, drug-induced renal failure, and chronic renal failure involve apoptosis. In particular, since an effective dose does not cause a decrease in blood pressure, it is also useful for patients and medical conditions that may be adversely affected by a decrease in blood pressure.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 25/28 A61P 25/28 43/00 43/00 (72) Inventor Kazutaka Hayashi Invited Otani, Hirakata City, Osaka Prefecture 2-25-1, Yoshitomi Pharmaceutical Co., Ltd. Drug Discovery Research Laboratory (72) Inventor Toshiaki Akira 2-25-1, Odaiya, Hirakata City, Osaka Prefecture Yoshitomi Pharmaceutical Co., Ltd. Drug Discovery Research Laboratory F-term (reference) AA02 DA05 MA01 MA03 MA04 MA24 MA43 NA06 NA14 ZA16 ZA81 ZC12 ZC41

Claims (1)

[Claims] 1. PGK ₁ , PGK ₂ , bicyclo PG
A cytoprotective agent comprising E ₂ , a salt thereof, or a derivative thereof as an active ingredient.