JPH09194376A - Adrenocortical hormone potentiating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent containing a specific phosphoric acid diester compound or its salt, potentiating an expression of a mediating gene for an adrenocortical hormone, also extremely low in toxicity, excellent in safety and useful for a potentiating agent for anti inflammantory actions, and for treating a dysadrenocorticism, etc. SOLUTION: This adrenocortical hormone potentiating agent contains a compound of the formula (R1 , R2 are each H or methyl) or its salt. This medicine can be prepared as a solid preparation such as tablets, granules, powder, capsules, etc., or a liquid preparation such as injections, oral liquid preparations, etc., and administered orally, or parenterally by intravenous injection, subcutaneous injection, intramuscular injection drip infusion, etc. Further, this medicine is administered preferably by 0.5-200mg, especially 2-50mg, based on the amount of the compound of the formula, once a day for an abult in the case of an injection, and by 5-2000mg, especially 20-500mg, as one dose and several times a day for an adult in the case of an oral preparation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a useful adrenocortical hormone enhancer. More specifically, the present invention comprises a phosphodiester compound of ascorbic acid and tocopherol or a pharmaceutically acceptable salt thereof,
The present invention relates to a corticosteroid enhancer.

[0002]

2. Description of the Related Art In the present specification, the term "adrenocortical hormone potentiating agent" refers to a corticosteroid-like potentiating agent which is expressed by a complex of a corticosteroid receptor and a ligand present at a target pharmacological action site. The adrenocortical hormone-like action refers to various pharmacological actions such as an anti-inflammatory action and an adrenocortical dysfunction treatment action which are expressed through intracellular adrenocortical hormone-specific receptors present throughout the body. The ligand refers to a compound that expresses a corticosteroid-like action such as corticosteroid and dexamethasone capable of binding to a corticosteroid receptor.

Conventionally, when a corticosteroid itself is used, serious side effects such as decreased glucose tolerance, exacerbation of infectious diseases, delayed wound healing, steroidal cataract, glaucoma, etc. occur, which is regarded as a very clinical problem. It is said that it is important to separate medicinal effects and side effects.

In addition, the adrenocortical hormone receptor is a member of the nuclear hormone receptor superfamily and is a ligand-inducible transcription factor, and by cloning the adrenocortical hormone receptor and other members of the family and analyzing the function of the nuclear receptor, The adrenocortical hormone receptor and other members of the family may also have a modular organization divided into three major functional domains, a C-terminal hormone binding domain, an N-terminal domain involved in transcriptional activation and a central DNA binding domain. Was revealed.

In addition, the adrenocortical hormone receptor c
It is known that DNA also contains two copies of a nuclear localization signal (NLS) for entry of the adrenocortical hormone receptor whose structure has been changed after ligand binding of an agonist into the nucleus.

On the other hand, the cytoplasmic heteromeric receptor complex is composed of an adrenocortical hormone receptor and various intracellular heat shock proteins (hsp), and in particular, the dissociation of 90 kilodalton (kDa) hsp is usually Is a prerequisite for the transport of ligand-bound receptors to the nucleus, and that corticosteroid-mediated signals cross-talk with many other signaling systems.
It was revealed what to do.

Therefore, in order to separate the efficacy and side effect of the corticosteroid-like action, it is necessary to avoid overdose of the drug having the corticosteroid-like action, and a prosthetic molecule that enhances the corticosteroid-like action is required. In particular, it is considered important to search for prosthetic molecules that enhance the expression of corticosteroid-mediated genes. However, at present, there is no drug that is a prosthetic group that is safe and enhances the expression of corticosteroid-mediated genes, and the advent of a corticosteroid enhancer has been awaited.

[0008]

In view of the present circumstances, the present inventors have made various studies on the mechanism so far in search of an excellent enhancer of corticosteroid-like action. As a result, the present inventors have found that suppression of the transcriptional regulatory function, which is an intermediary gene expression of adrenocortical hormone, is useful for enhancing the adrenocortical hormone action.
Furthermore, they have found that certain phosphodiester compounds effectively suppress the transcriptional regulatory function and can be expected as an adrenocortical hormone enhancer, and completed the present invention.

[0009]

That is, the present invention provides:
(1) The following formula

[0010]

Embedded image

(In the formula, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a methyl group.) Or a pharmaceutically acceptable salt thereof (hereinafter referred to as the present compound). Abbreviation), (2) an anti-inflammatory potentiator, (1) an adrenal cortex hormone enhancer, (3) an adrenocortical dysfunction treatment enhancer (1). The adrenocortical hormone enhancer described,
It is about.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The corticosteroid enhancer of the present invention can be applied as a corticosteroid potentiator having various actions such as anti-inflammatory action and adrenocortical dysfunction treatment action. The adrenocortical hormone enhancer of the present invention can be applied as an enhancer for any ligand that acts on the adrenocortical hormone receptor. This ligand may be either an adrenocortical hormone existing in the body or an exogenous adrenocortical hormone-like substance as long as it can bind to the adrenocortical hormone receptor.

The compound used as an active ingredient in the corticosteroid-enhancing agent of the present invention is, for example, Japanese Patent Publication No. 2-4.
It can be appropriately synthesized according to the methods described in JP-A No. 4478 and JP-A No. 62-205091, or in accordance with these.

The present compound used for the adrenocortical hormone enhancer of the present invention is an anti-cataract agent, a prophylactic / therapeutic agent for menopausal disorders, and a cosmetic having a skin beautifying effect (Japanese Patent Publication No.
No.), an anti-inflammatory agent (Japanese Patent Publication No. 1-27044), an anti-ulcer agent (Japanese Unexamined Patent Publication No. 63-270626), and a preventive / therapeutic agent for ischemic organ injury (Japanese Unexamined Patent Publication No. 2-111722). Already known. However, it has not yet been known that this compound is useful as an adrenocortical hormone enhancer.

The present compound used for the adrenocortical hormone enhancer of the present invention, whether it is a free compound or a pharmacologically acceptable salt thereof, is appropriately used for the purpose of the present invention. You can Examples of the pharmacologically acceptable salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and other salts. Any salt can be used as appropriate as long as it is a pharmacologically acceptable salt.

The adrenocortical hormone enhancer of the present invention may contain one kind or two or more kinds of the present compounds in an appropriate combination depending on the purpose and need.

The compound used as an active ingredient of the corticosteroid-enhancing agent of the present invention has extremely low toxicity and excellent safety, and therefore can be advantageously used for the purpose of the present invention [eg, the compound Among them, L-ascorbic acid, which is a typical compound, and DL-α-tocopherol phosphate diester potassium (a compound in which R ₁ is a methyl group and R ₂ is a methyl group in the formula [I], hereinafter EPC-
It is abbreviated as K. ) LD ₅₀ is 5g / kg by oral administration
(Rat), 100 mg / kg for intravenous injection (rat)
That is all.].

The adrenal cortex hormone enhancer of the present invention is, unless it is against the object of the present invention, a second adrenal cortex hormone enhancer other than the present compound, a corticosteroid hormone drug, or a component selected from another component having a pharmacological effect. Ingredients may be included.

The corticosteroid enhancer of the present invention is appropriately used orally or parenterally (eg, intravenous injection, subcutaneous injection, intramuscular injection, drip). As the form of the preparation, any of solid preparations such as tablets, granules, powders and capsules or liquid preparations such as injections and oral solutions can be appropriately prepared by known methods. For these formulations, commonly used excipients, binders, disintegrants,
Various additives such as dispersants, reabsorption promoters, buffers, surfactants, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers and pH adjusters may be used as appropriate.

The corticosteroid potentiator of the present invention can be advantageously used as a corticosteroid potentiator for mammals such as humans, dogs, rabbits, cows, horses, monkeys, cats and sheep.

When this compound is used as a corticosteroid-enhancing agent in, for example, humans, the dose varies depending on the type of compound used, the age, weight, sex of the patient, the indication, and the dosage form, and for example, injection. In the case of a drug, the dosage is about 0.5 to 200 mg once a day for adults, preferably about 2 to 50 mg.
In the case of oral administration, about 5 mg, several times a day for adults, about 5 ~
It is recommended to administer 2000 mg, preferably about 20 to 500 mg.

The present invention will be described in more detail with reference to Examples and Formulation Examples, and the effects of the present invention will be clarified by Test Examples, but the present invention is not limited thereto.

[0023]

【Example】

[Experimental example] [Experimental material and method] [1. Cells] are corticosteroid receptor-expressing Chinese hamster ovary cells (CHO-pM
T) was used. CHO-K1 cells are stably transfected with a full-length adrenocortical hormone receptor expression vector having a metallothionein gene promoter as a regulatory gene. The cells express a full-length corticosteroid receptor of 300,000 to 500,000 molecules / cell. The concentration of each cell
Ham's F- supplemented with antibiotics and 10% fetal bovine serum in the presence of 40 μM cadmium and zinc ions
The cells were maintained and cultured in 12 medium. One day before the experiment, the medium was replaced with Ham's F-12 medium supplemented with antibiotics and 10% fetal calf serum. In all experiments, steroids in serum were removed using dextran-coated charcoal and cells were cultured at 37 ° C. in O ₂ humidification in the presence of 5% CO ₂ .

[2. Plasmid] As the reporter construct, MTV containing chloramphenicol acetyltransferase (hereinafter abbreviated as MTV-CAT) and MTV not containing chloramphenicol acetyltransferase (hereinafter abbreviated as ΔMTV-CAT) were subjected to the experiment.
As the adrenocortical hormone receptor responsive substance-Luc, an adrenocortical hormone responsive luciferase expression reporter plasmid was used. The pCAT control vector is Promega (Madison, Wis.)
Obtained from. β-galactosidase expression plasmid p
CH110 (Pharmacia LKB, Uppsala, Sweden) was used as an internal control to know the transformation efficiency.

[3. Preparation of Whole Cell Extract and Partial Purification of Corticosteroid Receptor] CHO-pMT adrenal gland in 10 mM Tris-HCl (pH 7.8) buffer containing 1 mM ethylenediaminetetraacetic acid, 10% glycerin and 0.4 M sodium chloride. Cortical hormone receptor cells were homogenized. The homogenate was centrifuged at 100,000 xg for 1 hour at 4 ° C and the resulting supernatant was used as the crude whole cell extract. The partially purified adrenocortical hormone receptor was prepared from CHO-pMT adrenocortical hormone receptor whole cell extract. Whole cell extracts were prepared in the presence of molybdate and chromatographed on a phosphocellulose column. Next, the eluate was applied to a diethylaminoethyl (DEAE) cellulose column (Pharmacia LKB), and the adsorbed substance was eluted with 200 mM sodium chloride. From the pooled eluate,
Sephadex G-25 (Pharmacia LKB)
The salt and molybdate were removed by chromatography. After transformation (60 minutes at 25 ° C), the receptor fraction was further purified by protein liquid anion exchange Mono Q chromatography (Pharmacia LKB). Purified receptor-containing fractions were identified by ligand binding assay and specific DNA binding assay. These fractions contained 10-20% pure receptor. This receptor-containing fraction was designated as Protein-D.
It was used for experiments such as NA interaction.

[4. Hormone binding assay] Using a 6-well flat bottom plastic plate (manufactured by Corning), CH
O-pMT adrenocortical hormone receptor cells were cultured to confluence, and 6 hours before the hormone assay, the medium was Opti-MEM medium in the presence or absence (control) of 0.01-1 μMEPC-K. (Manufactured by Gibco). 2 with phosphate buffer isotonic buffer (hereinafter abbreviated as PBS)
After washing once with Opti-MEM medium (manufactured by Gibco), the cells were washed with 20 nM [ ³ H] dexamethasone (30-
50Ci / mmol; manufactured by NEN Laboratories) and incubated in the presence or absence of a 500-fold molar excess of non-radioactive dexamethasone (manufactured by Sigma) at 37 ° C. for 1 hour in 3 wells each. Wash monolayer 3 times with ice-cold PBS, 25 mM
CHAPS (3- 〔(3-Cholamidopropyl) dimethylammonio〕 -1-p
The cells were lysed in a solution consisting of ropane-sulfonate (manufactured by Sigma) and 1% sodium dodecylsulfonate (hereinafter abbreviated as SDS). A portion was added to scintillation fluid and the bound radioactivity was measured. The difference between total and nonspecific binding was considered specific binding and was used as the number of corticosteroid receptors in this study. As a result, 0.01 ~ 1 μM E
It was found that addition of PC-K does not affect the ligand-binding activity of the adrenocortical hormone receptor.

[5. Western immunoblot analysis] The cytosol was fractionated by 10% SDS-polyacrylamide gel. After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane (Bio-Rad, Richmond, Del.) And a rabbit was immunized with a synthetic peptide that aligned the amino acid residues between human adrenocortical hormone receptors 346 to 367. Anti-human antibody PA generated there after
Probing was performed using 1-512 (Affinity Bioli Agent, Nashenic Station, NJ). Antigen-antibody complex, Prot Blot
It was detected by a system (manufactured by Promega Corp.) according to a conventional method. As a result, it was found that the addition of 0.01 to 1 μM EPC-K did not affect the human adrenocortical hormone receptor level.

[6. Electrophoretic mobility shift assay (hereinafter abbreviated as EMSA)] A double-stranded oligonucleotide probe was prepared using Klenow fragment (DNA polymerase I large fragment) of DNA polymerase I (Takara, Japan) [α- ³² P] dCTP (manufactured by Amersham) was end-labeled, and unincorporated nucleotides were removed by chromatography using a Nick column (manufactured by Pharmacia). Partially purified adrenocortical hormone receptor (usually 10 ng protein per reaction)
[ ³² P] end-labeled corticosteroid receptor response factor probe oligonucleotide (about 20,000 cpm) 0.2 ng together with 5 mM HEPES, pH 7.9, 60 mM potassium chloride, 2.5 m
Incubated on ice for 15 minutes in a 20 μl reaction mixture containing MEDTA, 2.5 mM magnesium chloride, 10 mM spermidine, 0.25 mM dithiothreitol, 10% glycerin and 100 ng poly (dI-dC) (Pharmacia LKB). Non-radioactive D
NA included. The reaction mixture was then loaded on a 4% non-denaturing polyacrylamide gel containing 1/4 diluted TBE (TBE is 89 mM tris-borate, 89 mM boric acid and 2 mM ethylenediaminetetraacetic acid in water). . The gel was electrophoresed at 350 V for 2 hours and dried. The results were visualized by autoradiography. Also,
The sequence of the oligonucleotide containing a corticosteroid response factor (corticosteroid receptor response factor) is 5 '.
-CGAGTAGCAGACACAGGATGATCT
GAGG-3 '. For quantification, the BAS2000 phosphor image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan) was used to measure the radioactivity in the appropriate band. As a result, addition of 0.01 to 1 μM of EPC-K resulted in a decrease in EPC-K relative to (amount of corticosteroid receptor-adrenocortical hormone receptor response factor complex formation).
Was found to increase depending on the concentration.

[7. Transfection and assay of chloramphenicol acetyltransferase and luciferase (hereinafter sometimes abbreviated as CAT)]
Transient transformation (trnscient steroid transfection)
Was done. Briefly, cells are plated in a plastic culture dish with a diameter of 10 cm until 30% confluent and then PBS.
It was washed 3 times with, and then the medium was replaced with Opti-MEM medium. The plasmid cocktail was added to Lipofecti (Lipofecti
n) A reagent (manufactured by Gibco) was mixed and added to the medium. After a 12 hour incubation, the medium was replaced with fresh medium containing 2% FCS stripped with dextran-coated charcoal and the cells were cultured for an additional 24 hours in the presence or absence of various ligands. After standardizing the transfection efficiency by the expression of β-galactosidase, the radioactivity of the corresponding spot was measured using a BAS2000 fluorescence image analyzer (Fuji Photo Film Co., Ltd.) for quantifying the enzymatic activities of CAT and luciferase. Measured. Chemiluminescence is LB9501 luminometer
(Manufactured by Bell Salt Co., Ltd., Japan, Tokyo).
The results are shown in FIG. As shown in FIG. 1, EPC-K enhanced dexamethasone-induced CAT expression in a concentration-dependent manner.

From the above results, it was found that the treatment of cells with EPC-K did not affect the level of adrenocortical hormone receptor protein, the amount of the complex formed with adrenocortical hormone receptor and adrenocortical hormone receptor response factor complex was increased, and the pCAT control vector was used. EPC for adrenocortical hormone-induced gene expression without affecting expression
The positive effect of -K was revealed to be specific for corticosteroid receptor and corticosteroid receptor response factor interactions. From the above, it was found that EPC-K is useful as a corticosteroid hormone enhancer.

[Formulation Example 1] Oral tablet EPC-K 100 mg Lactose 75 mg Starch 20 mg Polyethylene glycol 6000 5 mg The above ingredients are admixed by a conventional method to give a tablet for 1 tablet.
A sugar coating may be added if necessary.

[Formulation Example 2] Injection EPC-K 200 mg Mannitol 5.0 g 1N-sodium hydroxide proper amount distilled water total amount 100 ml pH 6.5 The above components are dissolved by a conventional method and sterile filtered. The filtrate is aseptically filled into glass ampoules in an amount of 5 ml each, and the mixture is sealed to obtain an injection.

[0033]

INDUSTRIAL APPLICABILITY The preparation of the present invention enhances the expression of corticosteroid-mediated genes, and is therefore useful as a corticosteroid enhancer.

[0034]

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of EPC-K on adrenocortical hormone receptor transcription activation function.

Claims (3)

[Claims] 1. The following formula: (In the formula, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a methyl group.) A corticosteroid potentiator comprising a phosphodiester compound represented by the formula or a pharmaceutically acceptable salt thereof Agent. 2. The adrenocortical hormone enhancer according to claim 1, which is an anti-inflammatory enhancer. 3. The adrenocortical hormone enhancer according to claim 1, which is a therapeutic enhancer for adrenocortical dysfunction.